Advances in DNA Sequencing Technologies

Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t Advances in DNA Sequencing Technologies Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t

Faust Essay -- essays research papers

The legend of Faust was a legend that occurred in the 1500’s in Europe. Over time, as the story was told and passed on through generations, many different ideas on what happened were brought up, but the main idea of the story is the same in most cases. One of the most interesting things about this legend is the fact that though this story is more than four hundred years old, it is still told in some contemporary films to this day. All though it is not always as direct as a deal with the actual devil, the same basis of the story can be seen in present day films. In one of the most successful movies of the year 2000, The Matrix, a Faustian theme is evident. The Matrix is a science fiction movie directed by the Wachowski brothers. The old legend of Faust is, in short, about a young scholar who made a deal with Mephistopheles, the devil. Faust was seeking ultimate knowledge and in the deal the devil said he would grant Faust ultimate knowledge in return for his soul. Faust agrees to the deal and after a certain time period of possessing ultimate knowledge Faust suddenly dies. There are many different versions of the story as to exactly how he died, and some versions of the story go into more detail than others. As time passed, Faustian legends were being told in many different stories, many different ways. To have a Faust story, four basic elements should be present: a Faust figure, a devil figure, some sort of temptation, and a price.   Ã‚  Ã‚  Ã‚  Ã‚  The Matrix is about a man, called Neo, who was living an average life, and was heavily into computer hacking. One day he receives messages appearing on his computer leading him towards a meeting with a powerful man named Morphius. Morphius alerts Neo that the reason that the reason that all of federal agents were chasing him and all of these other things were happening to him because he was â€Å"the one†. He was searches for a greater truth in the world than what was just there in his face, and Morphius says that he could show Neo that truth. Morphius then holds two pills in his hands, one pill would lead him to the truth, the other would just take him back to his regular life as if nothing ever happened. Neo wanted the truth about the world so Morphius explained it. He said that the perception is that our day-in, day-out world is real; in reality, that world is a hoax, an elaborate deception spun by al... ...ant to make Faust a hero of some sort.   Ã‚  Ã‚  Ã‚  Ã‚  If someone could go back in time and show this film to people that know of the Faust legend back in around the 1700’s, it would make absolutely no sense. It would seem like a whole lot of unrealistic garbage to those people, but in present times, though the Matrix is very futuristic, it almost seems as if it could be possible. The whole idea of the movie plot seems brilliant and makes people question themselves to whether or not this concept could actually be possible.   Ã‚  Ã‚  Ã‚  Ã‚  The links between the Matrix and the Faust legend can be seen throughout the entire film. Even though the storyline of the legend has gone through a number of changes over time, it is truly amazing how a story that probably is not even true still comes into use in society today. The Faust legend is used in other situations besides just films, stories, or plays; it is also seen in everyday situations for people. There are many ways in which a Faustian theme can come into play in everyday life. For example, any one who has ever sold themselves out for something, gambled, or done drugs has been in a Faustian situation.

E-Procurement And E-Logistics

our site – CUSTOM ESSAY WRITING – Business Management Dissertation Ideas ABSTRACT In this paper, we analyze the e-procurement and e-logistics of the Dell Inc. Company. This will include a brief overview of the company, an exploration of its Customer Relationship Management, the Supply Chain management and an analysis of the various softwares used by Dell Inc in promoting its relationship marketing. INTRODUCTION Today, many people have discovered the significance of E-commerce. E-commerce, also known as electronic commerce refers to business transactions and communication via computers especially over the internet and networks (Botha, Bothma and Geldenhuys, 2008: p.23). This involves buying and selling of services and goods, and transfer of funds among other commercial communications through the internet, mainly through the World-Wide Web (Botha, Bothma and Geldenhuys, 2008: p.23). E-commerce takes place in different situations such as between businesses and customers (B2C), between one business/company and another (B2B), and between customer and customer (C2C). It is mainly divided into two main parts, which are e-procurement and e-logistics. E-procurement is defined as an electronic method of conducting business transactions while e-logistics refers to the transfer of goods sold over the internet to customers (Botha, Bothma and Geldenhuys, 2008: p.24). A well implemented e-procurement system is highly effective in connecting businesses and other business processes with suppliers while running all interactions between them. According to Botha, Bothma and Geldenhuys (2008: p.23), the development and advancement of technology, many businesses now sell their products through computer technology, which is a brilliant way of making companies reduce overhead costs and reach a wide customer base. Thus, e-procurement benefits not only the business owners, but also customers since they can shop without leaving their homes. Also, customers can easily find the lowest price of products when buying their goods via the internet. In this paper, we analyze the e-procurement and e-logistics of the Dell Inc. Company. DELL INCORPORATED Dell Inc. is a computer company that was established by Michael S. Dell, in 1984 (Krauss 2003: p.7). It offers a wide range of technology product categories (Krauss (2003: p.8). These products range from personal computers to services such as storage solutions. Also, it gives a variety of services, which range from business services and configurable information technology including product-related support services, consulting and applications and infrastructure technology (Krauss, 2003: p.8). As stated by Levy (1999: p.20), Dell Inc. operates in four global business segments, which include public, Large Enterprise, Consumer, and small and Medium Business. The company designs its own products, manufactures and markets them, sells, as well as supports a range of products and services, which can be modified to individual requirements of customers (Perret and Jaffeux, 2007: p.4). Dell Inc. is considered among the companies that are most profitable. The company offers the most innovative customer service, as well as product custom configuration in the world (Perret and Jaffeux, 2007: p.5). For this reason, the company is faced with the challenge of satisfying the customers’ needs while maintaining a stable relationship with them. E-PROCUREMENT AT DELL Dell Inc. is widely known for selling its computers and others services through the internet to other business (B2B) and to individual customers (B2C) (Perret and Jaffeux, 2007: p.5). B2B refers to business transactions between one company and another such as business customers, suppliers and distributors. The B2C refers to business transactions between a company and consumers. At the beginning of the 1990’s, Dell Inc. attempted to distribute wares by retailing. However, the management found out later that this method was unprofitable for business (Gattorna, 2003: p.51). Hence, Dell Inc. decided to key on boosting its customer support and services by allowing customers to make orders directly (Gattorna, 2003: p.52). This was considered a unique strategy for Dell customization. Recently, Dell Inc. improved its sourcing and buying processes by implementing a leading e-procurement solution known as Ariba Buyer (Krauss, 2003: p.8). In order to ease the business processes between Dell Inc. and its supplier companies, Ariba Buyer which is an e-procurement solution is used. It is quite useful in automating and streamlining sourcing. (Li, 2007: p.20). In earlier years, making purchase orders at Dell was a highly laborious process since company workers filled out forms for each purchase process every time they ordered an item, which included collecting about ten approval signatures (Li, 2007: p.21). The buyers were then expected to re-enter the data into two different systems that included a home-grown Access database and the legacy purchasing system. This paper-based process was challenging for Dell to track its purchases by commodity, as well as analyze its purchasing patterns in terms of where, how much and from whom the supplies were bought, hence the change in its procurement process. Thus, Dell Inc. implemented an e-procurement solution known as Ariba Buyer. E-procurement enabled Dell to streamline its supplying base. This helped in the elimination of maverick spending, as well as standardization of the ordering processes for its suppliers. (Krauss, 2003: p.8). This was followed, by Dell’s move, to assess 3 e-procurement systems depending on five criteria. These criteria included a user-friendly boundary, cost-effectiveness, and integration with existing back-end system (Krauss, 2003: p.8). Others included e-commerce links to most of Dell’s supplying companies, and compatibility with the current IT policy of Dell servers (Li, 2007: p.20). According to Gattorna (2003: p.50), close to seven months were spent by the personnel that were responsible for implementing Ariba. This time was spent in developing twenty interfaces that would facilitate connection of Ariba buyer with Dell’s legacy systems. They created linkages for Ariba and Dell’s purchase order, catalog data, cost center, accounting code validation, and employee data among other systems (Gattorna, 2003: p.50). This was made to ascertain that all the processed orders had been validated. This resulted in a final product, which facilitates making purchases online. This product is known as Dell Internet Requisition Tool (DIREQT) (Gattorna, 2003: p.51). Currently, DIREQT has made it easy for Dell employees to complete purchasing orders online by loging into DIREQT Web site, as well as conducting searches for certain products, suppliers or services, which usually give accurate status reports (Levy, 1999: p.23). Immediately, Ariba Buyer forwards the catalog items and requisition straight to the right manager at the cost center who signs the order electronically. The system then automatically creates an approving chain before directing it to an employee network. (Gattorna, 2003: p.51). However, if the product ordered is not present in the catalogue, Ariba Buyer includes a Dell buyer to source the product and hands over the request for last signatures (Perret and Jaffeux, 2007: p.6). After the requisition has been approved, it is moved to the Ariba Commerce Services Network (ASCN). ASCN is a shared network infrastructure that helps to connect with buyers and marketplaces, on the Ariba Business to Business (B2B). Commerce stand (Perret and Jaffeux, 2007: p.6). Ariba uses ASCN to communicate its orders to suppliers, which includes shipping through e-mail, faxes, Extensive Markup Language (XML) and electronic data interchange (EDI) (Perret and Jaffeux, 2007: p.6). Moreover, Ariba Buyer also accelerates the payment process in Dell Inc. The receipts that Dell’s central receiving department prepares for wares are brought into the organization and matched automatically with the right invoice. This is then fed into the system by the account payable processors (Bothma and Geldenhuys, 2008: p.25). In addition, the purchasers create receipts of the service given to them, which is also matched in an automatic manner. Therefore, this practice helps to avoid the early routine of service invoices, which is time-consuming, when making purchases for approval. As stated by Botha, Bothma and Geldenhuys (2008: p.25), with the Ariba Buyer at Dell, the requisition cycle time is likely to be reduced by 62%, and lessen operation costs by 61%. However, Dell Inc. believes that it stands to benefit on a larger scale from the perception into the buying process attained through combining customers’ information. Moreover, through the use of Ariba, Dell is able to gather information necessary to evaluate its supply base and re-evaluate key business to market communications services, office products and consulting, among many more kinds of expenditures (Gattorna, 2003: p.50). CUSTOMER RELATIONSHIP MANAGEMENT According to Perret and Jaffeux (2007: p.7), Customer Relationship Management (CRM) is the creation and maintenance of relations with customers. The key aim of Dell is to offer its customers technologically reliable customer service requirements. Perret and Jaffeux (2007: p.7) argue that the software that help in facilitation facilitate Dell’s CRM include marketing automation software, a system that benefits the sales, and custom designed Web pages that contain purchase data. According to Ross (2010: p.88), today, one fifth of standard-based computers sold in the world is Dell’s product. The key concept of Dell Inc. is to sell computers directly to customers. This will increase their success in the computer business. (Ross, 2010: p.88). Before Dell Inc. invented the made-to-order concept, its customers used to buy its products from electronic shops and retail stores. In this case, customers interacted only with the salesperson of the store and not the manufacturer. Therefore, Dell introduced the concept of interacting directly with the customer via the internet so as to fulfil the demands of its clients and deliver quality services. E-LOGISTICS AT DELL INC. For Dell Inc., the E-logistics has entirely changed it way of distributing its products. Traditionally, Dell used to pick up components from the warehouses of suppliers then collect them in its central or regional distribution centres, and finally merge them in stock in order to deliver the final products to customers (Ross, 2010: p.88). Currently, through implementation of e-logistics, Dell Inc. can now pick up components from the ware houses of suppliers and then forward the merging of components made during the transit to the logistic-service providers through USP or Airborne Express (Li, 2007: p.36). This has resulted in less fixed costs spent in warehouse centers and distribution, no product technological obsolescence, and no stock-keeping units (SKU). SUPPLY CHAIN MANAGEMENT AT DELL INC. Supply Chain Management (SCM) is a system that Dell established to ensure the availability of precise computer components for its customers on demand and location. SCM describes how the company manages how raw materials are transformed into the end products and how products and services get to all its consumers (William, 2003: p.150). This has enabled the company to develop a tight bond with its supplier companies and consumers. In this regard, Mencarini (2003: p.19) states that Dell Inc. have one of the most effective SCM system in the world, and that it is focusing on creating the best SCM through the i2. This will improve the supply chain process through connecting its suppliers and planners in order to satisfy the requirements, as well as demands of their customers. SOFTWARE USED BY DELL IN PROMOTING RELATIONSHIP MARKETING Dell also uses a variety of software to promote relationship marketing such as Hotlink, Premier Pages and an enhanced CRM system, among others (Gattorna, 2003: p.57). Its database software is highly efficient and effective with customer relationship management, which stores tables of data used to check the information of customers and establish promotional campaigns. These databases mainly include the information of customers, their products and interests. According to Gattorna (2003: p.57), customer database helps to increase profits since it contains the information of clients, which determines the efficient and effective ways to target and divide the consumers. Hotlink is an automation software program, which facilitates tergeting and marketing communication, monitoring of customers and market development (Mencarini, 2003: p.21). This software gives Dell a free opportunity to advertise its products through the word of mouth. Also, it impacts its customer base to ensure that customers receive better services than before. Premier Pages are a transparent online system/software custom designed Web pages, which contain all the purchasing data (Gattorna, 2003: p.57). In addition, the software contains a paperless ordering process, which captures the technology configurations of customers. Mencarini (2003: p.21) argues that Dell created Premier Pages in order to gather less clientele details than they already have and develop a win-win situation that is more realistic. This starts when the clients places their orders for a computer and built later. Another system that Dell uses is an enhanced CRM system, helped by an information system company called the IS Partners (Moon, 2003: p.45). ProClarity offers a comprehensive analytical ability that highlights negative and positive areas of the business. Moreover, the company breaks down its sales by region where each team enables Dell to measure its own trend and success. ProClarity significantly benefits all the financial sections of the company. It also helps the Dell staff to easily access detailed demographic information about customers. The marketing department is able to follow product sales, customer activity and marketing mixes via this software. The management can follow activities in customer accounts, and act on lapsed quotes. Additionally, Dell installed the e-commerce software i2 Collaboration Planner, i2 Supply Chain Planner and i2 Factory Planner in order to meet its supply chain needs (Moon, 2003: p.45). This is applicable in the management of build-order procedures that exist between placing orders and customer support. The software enables Dell Inc. to classify customers and target them through their most preferred medium, obtain and analyze results (Moon, 2003: p.45). Moreover, Dell Inc. has signed an agreement with Part-Miner (Gattorna, 2003: p.51). Part-Miner is a vertical portal in electronic components industry, which provides information and helps to meet the demand and supply of the components. FUTURE PLANS OF DELL INC In future, Dell plans to update its processes of purchase such as the establishment of online auctions for products and services like printing, shipping, and paper (Li, 2007: p.20). The company also plans to make order status, payment information and receipts easily accessible to suppliers online. In coming years, Dell intends to expand its catalogue base and purchase choices by convincing its main suppliers to use the Ariba Business to Business Commerce Platform (Li, 2007: p.20). CONCLUSION CRM-SCM integration tries to satisfy clients through prompt delivery of products, ensuring its accessibility and maintain the manufacturer’s profits and returns. Thus, there are several lessons that can be drawn from Dell’s application of e-business. This trend can be emulated by other organizations in the industry. This will result in offering of better services to customers. It can be portrayed via the way Dell Inc. uses CRM to its advantage. Customer satisfaction will increase their trust in the organization, improving its reputation. In addition, custom-building a PC desired by the clients has formed a particularly strong relationship between Dell and its customers (Moon, 2003: p.50). In addition to this, implementing technology in a phased fashion has helped Dell to achieve a strong relationship with its clients. Dell set up simulated environments in order to support the i2 system in blotches without affecting the live form. Dell ensured that all stages of the comp leted process allowed future growth of the company before developing the whole system. Hence, this reduced risk and increasing efficiency. Another significant lesson from Dell would be to extend the link from the customer to the supplier, while maximizing its operation efficiency as well as customer satisfaction (Ross,2010: p.92). As a result, customers were able to spend less money on purchasing customized machines. This is because Dell approved the savings that resulted from managing its inventories efficiently. The company was, therefore, able to share information with suppliers about customer requirements and buying patterns in real-time. REFERENCES Botha, J., Bothma, C. & Geldenhuys, P. 2008. Managing E-commerce in Business, New York, Juta and Company Ltd. Gattorna, J. 2003. Gower handbook of supply chain management, Burlington, Gower Publishing Ltd. Krauss, M. 2003. Dell looks to Sears to extend buyer reach. Marketing News, April 28, 2003, Vol. 37, Issue 9. Li. L. 2007. Supply chain management: concepts, techniques and practices enhancing the value through collaboration, Tokyo,World Scientific. Moon, K. 2003. Dell Computers: A Leader in CRM. Retrieved February 20, 2010 Mencarini A. 2003. E-Business: Dell Case Study, UK, Strathclyde Business School. Perret, F. & Jaffeux, C. 2007. Essentials of logistics and management, London, EPFL Press. Levy, R. H. 1999. The Visible Marketer: Dell’s CRM model stresses transparent processes. Available from http://directmag.com/mag/marketing_visible_marketer_dells/index.html {Accessed 20th February 2012} Ross, D. F. 2010. Introduction to Supply Chain Management Technologies. London, CRC Press. William C. 2003. The true meaning of supply chain management. Logistics Management, June 2003, Vol. 42, Issue 6.

Distribution Channel Management Essay

BMW Motorcycles is a division within the BMW Group. As such their mission is tied to that of the parent company. On the BMW Group website it is noted as follows:†Identifying potential and encouraging growth. Knowing what we represent. Recognizing where our strengths lie and making the best use of every opportunity. Following a clear strategy. Goals we have attained are in essence the point of departure for new challenges. This is the philosophy that inspires every individual at the BMW Group. It influences the company’s structure and it plays a vital role in the decision-making process. Our corporate ethos finds its expression in the uncompromising pursuit of the superlative. The result? Outstanding brands with an unmistakable profile. Automobiles and motorcycles which fascinate people all over the world and which win legions of new admirers every day. And a degree of success which sees the BMW Group go from strength to strength. With the three brands, BMW, MINI and Rolls-Royce Motor Cars, the BMW Group has its sights set firmly on the premium sector of the international automobile market. To achieve its aims, the company knows how to deploy its strengths with an efficiency that is unmatched in the automotive industry. From research and development to sales and marketing, BMW Group is committed to the very highest in quality for all its products and services. The company’s phenomenal success is proof of this strategy’s correctness.†BMW Motorcycles provides a robust product line following three general categories: Touring, Sport, and Enduro. Touring bikes are built for the long haul, with rider comfort as the premium. These are the motorcycles you would take on a cross country trip. Sport bikes satisfy the â€Å"need for speed† and styling trends for the younger crowd. Enduro bikes are for the adventurer is us all; these bikes are built for on or off road travel. BMW Motorcycles’ main competition comes from Japanese motorcycle companies. Most American motorcycle companies produce cruiser style bikes; BMW offers little choice in this style of motorcycle. However, each of the main Japanese manufacturers (Suzuki, Yamaha, Honda, and Kawasaki) offers motorcycles that are very similar in style and performance. Although BMW has  historically had its own niche in the market, that of the older male, it has recently branched off into more dedicated off-road and super-sport bikes. These are categories that have been historically dominated by the Japanese manufacturers. Throughout the years, BMW motorcycles have been renowned for their durability and engineering. BMW has always been on the forefront of motorcycle engineering. BMW was the first motorcycle manufacturer to offer ABS on a motorcycle. They have also revolutionized motorcycle suspensions with their Telelever anti-dive suspension and Paralever single-arm suspension. Although BMWs are more costly, that price is offset by the life cycle of the motorcycle. Until recent years there hasn’t been much of a rivalry between the Japanese manufacturers and BMW Motorcycles. BMW had its small place in the market and was not a threat to the Japanese firms. BMW Motorcycles were not known to be stylish or fast, the two categories that attract the majority of motorcycle buyers. BMW attracted more mature owners who were impressed by the machine’s reliability and durability, and who had more money to spend on a new bike. The lack of power was partly due to BMW self-imposed limitations on horsepower in motorcycles. However, these limitations were lifted in the late 1990s, and BMW began to produce super-sport bikes that rivaled the Japanese bikes, and even surpassed the Japanese competitor’s performance. Up to this point, young males were rarely interested in purchasing BMWs. They cost twice as much as the closet Japanese bikes, they were slower, and they were less attractive. In essence they were the Volvo of the motorcycle world. However, like Volvo, once BMW redesigned their product line into more powerful and stylish motorcycles, they began to quickly cut into the Japanese manufacturers’ market share. BMW motorcycles have consistently won â€Å"Best Bike† awards in various categories within the past few years. Several of these awards include â€Å"2005 & 2006 Best Touring Bike† for the R1200RT, and â€Å"2005 Best Adventure Bike† for the F650GS. BMW Motorcycles reported December 2006 sales up 36.8% over the same period in 2005. In 2005, annual sales worldwide for BMW topped  100,000 motorcycles. In 2004, approximately 5.7 million motorcycles were sold in the US, of which 12,825 were BMWs. Estimates of market share for 2005 show Honda in the lead with 24% of the market, followed by Harley-Davidson and Buell with 22.6%, Yamaha at 15.9%, Suzuki at 11.8%, Kawasaki at 9.1%, KTM at 1.7%, BMW with 1.2% and â€Å"Other† at 13.7%. Although BMW gains less than 2% of total US motorcycle sales market, they only reflect less than half of BMW’s worldwide sales. BMW sold more motorcycles in Italy (13,651 bikes), than in the US, and Spain was a close third wit h 10,002 bikes. The BMW R1200GS has been the top selling bike worldwide for the past several years. As a result, Japanese motorcycle manufacturers have recently introduced large engine dual sport bikes to their lineups to compete with the BMW. The BMW R1200RT has been increasingly sought after by police departments as their bike of choice, replacing the mainstay Kawasakis. With their ever increasing popularity and sales, BMW is becoming a threat to the Japanese manufacturers and the rivalry is growing. Two of the primary attributes for motorcycles are durability and performance. Durability is defined as the capability of withstanding wear and tear, it is measured by a products ability to perform or compete over a long period. For a motorcycle this can include engine life, the ability to conduct repairs, and retention of market value. Performance is defined as the way in which someone or something functions. For motorcycles it includes horsepower, acceleration, cornering ability, and maximum speed. Target MarketsTarget Market 1: Young Males 18-25- Generally college age males, style and performance are the biggest factors for this group. Cost could be an exclusionary criterion, as this group may not have a large cash base. Target Market 2: Adult Males (25-40)- Established professionals. Performance not as big a factor as durability or economy is. This group is willing to spend more for a higher quality product. They are style conscious, but style is not necessarily a prime factor. Target Market 3: Females (all ages)- Female motorcycle buyers are a growing market, but still small enough to treat all ages as a singular group. Female riders generally are looking for something economical and stylish. Performance is low on the list of criteria. Primary Product Categories. -Enduro . These are dual purpose motorcycles that are built for both on and off road. -High Performance. Specialized off road motorcycle. -Tour. These are bikes built for long distance rides. Comfort is number one on these bikes. -Sport. These are your speed bikes. They are built to go fast and look good. -Urban. These are the bikes built to cruise the road and get good gas mileage while they are at it. This is the commuter bike category, not flashy but dependable. EnduroHigh PerformanceTouringSportUrbanYoung Male21 / 3Adult Male11 / 3Female221Core products2Rarely sold to these individuals3Biggest purchaser of items in this categoryImportant Cells:Young Male /Sport. This is the primary product line for this target market. They are looking for style and speed. This is the category that provides those. This line is very important, as this is the largest share of the entire market. BMW has been behind in this line, and only recently become competitive within the past few years. Although they now have one of the highest performing motorcycles, it is difficult to overcome the brand recognition the Japanese motorcycles have built up in this line. The competitive advantage in this line is gained through a mix of performance and appeal, with cost also a major factor. Perceived performance and style may be more important than true performance and style. Young Male / Urban. This is a growing market the past few years. BMW is neck and neck with all its competitors to grab a share, due to the newness of this motorcycle category. This is important, because getting the recognition and sales in this product line can affect the sales of the other lines. No company currently has an advantage. The advantage can be achieved by offering a motorcycle with excellent handling, great gas mileage, at a low cost. One of BMW’s products, the F650, has been seeing robust sales due to the high MPG. BMW is poised to gain an advantage in this market, the key will be to ensure the BMW name recognition is realized within this target market. Adult Male Enduro. BMW has had the leading product in this market since the introduction of their R – GS line of bikes. This award winning product has been continually recognized and awarded for its attributes. It is the king of the hill of enduro bikes, with no true competition in sight. This bike has become on of the cornerstones of BMW motorcycles, and thus its continued success will be a direct reflection of the company. BMW has been able to sustain their advantage in this market / line by continually improving the product. Adult Male / Touring. This category has seen large growth in the past few years. BMW is poised to gain an advantage here, due in large part to the recognition they are gaining by the police forces. Police forces have been buying their R-RT models in increasing numbers for the past five years. This has had a direct impact on the sales volume of not only this product, but the touring line as a whole. The police usage has given BMW greater brand recognition. Keeping the bikes visible within police forces will be the key to maintaining their competitive advantage. There are only two real competitors in this category, the Kawasaki Concours and the Honda Goldwing. The Honda has been the leader in this line until several years ago when BMW sales began to overtake them. BMW provides better durability, performance and comfort than the competitors. These are all key attributes for this target market. BMW has been pursuing new markets and products; they also have opportunity to develop both, especially in the female rider market. In the past few years, BMW has introduced improved models of their existing motorcycles to increase their appeal to younger markets. They have greatly increased their performance and styling, both key attributes sought after by this largest target market. As a result of developing this market, profits in the young male market have soared. In addition, BMW has introduced all new models of motorcycles. Of these new models, the most notable are high performance sport bikes to appeal to the young males, and less powerful, smaller frame bikes to increase appeal to beginners and females. Since 1999 BMW has developed close to 15 new motorcycle products for introduction. Each of these bikes has seen brisk sales. BMW does not hold a competitive advantage in the motorcycle market; this is due to several factors. BMW motorcycles are expensive. In many cases they cost more than $10,000 more than their market competitor. Although BMWs are more durable than their less expensive competition, the prime market does not appear to be willing to spend the increased cost for a higher quality product. In motorcycle sales, no company holds a sustained competitive advantage. The Japanese motorcycle companies, with their powerful, inexpensive motorcycles share the competitive advantage over the American and European motorcycle companies. However, this advantage tends to shift to a different company each year as new bikes are introduced. The price points for Honda, Yamaha, Kawasaki, and Suzuki are all similar for each comparable product; with generally only several hundred dollars difference. Any competitive advantage among these companies will rarely last over a model year or two. Thus there is no sustained competitive advantage among these rivals. Due to the similarities in the product offerings between all these companies, unless some revolutionary technology is developed, it will be nearly impossible to sustain any competitive advantage. Customer satisfaction is a high priority with BMW; in 2001 BMW Motorcycles introduced the â€Å"Customer-Oriented Sales and Production Process†. This process is used from customer order to delivery. It begins with an online  ordering system which allows a BMW Motorcycle dealer to send a customer’s order directly to the manufacturing plant, eliminating wholesalers or resellers and providing instant delivery status. This system also allows the customer to change any detail of his order, up to the minute that production is begun. All BMW motorcycles and parts are built at the BMW plant in Berlin, Germany. BMW motorcycles were originally built in BMW’s historic Munich plant until the 1960s. BMW concentrates all motorcycle related production, to include many component parts in the Berlin plant. This plant can build up to 500 motorcycles a day. BMW motorcycles are built to general specification and also may be custom designed from anywhere in the world. The Berlin plant builds motorcycles to the specific requirements of the over 20 countries they deliver to. The BMW motorcycle plant in Berlin uses state-of-the-art technology. It has been recognized as the most modern motorcycle manufacturing plant in the world. Like their motorcycles, the production plant is focused on ergonomics and efficiency. In addition, the factory incorporates numerous environmental friendly processes. Many of these processes, including production water recycling, also help to cut production costs. The BMW logistics division is responsible for organizing the motorcycle production. Together with marketing, sales and production departments it plans the production schedule, generates the best production sequence and ensures timely delivery of the motorcycle to the customer. Logistic specialists plan and coordinate the delivery of material to assembly and make sure that quality parts are cost-efficiently delivered to the production line. BMW Motorcycles has more than 400 external suppliers provide some 9,000 different parts and components. These parts are delivered to BMW’s Berlin plant on time and in the correct quantities. Most of the suppliers specialize in motorcycle parts for BMW. 65 % of the suppliers are located in Germany, 34 % in Europe and 1% in USA and Japan. In 2001, BMW reengineered its distribution process to reduce costs and improve response time for dealers and parts vendors. Once the motorcycle is completely assembled, it is transported from the factory direct to the retailer who placed the customer’s order through a â€Å"Retailer Channel† method. BMW uses a variety of transportation methods including truck, rail, and sea lift ; depending on the destination of the product. In the USA, BMW motorcycles are received and distributed out of two warehouse locations in New Jersey and California. From there, they are delivered direct to the retailer. BMW Motorcycle dealers are authorized by BMW to sell the motorcycle product line, and to also provide an after sales service to BMW customers. To maintain brand image and ensure customer satisfaction is met, the dealers comply with corporate requirements and guidelines in the presentation of their facility. BMW considers corporate identity and brand image to be paramount to maximize awareness of the differences between it and its competitors. These guidelines establish standards for interior and exterior design, levels of customer service, signage, typefaces and advertising. The goal of these requirements is to maintain BMW’s prestigious reputation. In addition, BMW provides management support, training courses for all dealer staff, recruitment assistance, marketing support, and an internal comprehensive online reference system which gives immediate access to up-to-date product and corporate information. BMW’s dedication to customer service, both service visible to the consumer and services which are transparent, give it a decisive advantage within its distribution channels. By striving to provide the customer the exact product desired, in a timely manner, they have streamlined their distribution processes. In addition to providing satisfaction to the consumer, this retailer channel process has cut transportation and resale costs to the benefit of both BMW and the customer. The benefits of BMW’s â€Å"Customer-Oriented Sales and Production Process† are  that the consumer can get an individualized BMW motorcycle without having to purchase a cookie-cutter motorcycle and numerous after-market products. The customer gets his one of a kind motorcycle right off the production line. In addition, he can order his customized motorcycle and receive it in a comparatively short period of time, with instantaneous production progress status available upon request. These are benefits to the consumer that the competitors cannot provide. With Japanese manufacturers, they sell the base motorcycle with very limited options. It is a dice roll that the configuration you want is in stock locally. This level of customer service, along with the high quality of the motorcycles, creates a rabid dedication to the BMW brand. BMW motorcycle owner typically will purchase another BMW over any of the competitor’s brands. This is decidedly one of the factors giving to the steady increase in market share of BMWs, and thus proof of BMW’s competitive advantage. References1. BMW Group. http://www.bmwgroup.com2. BMW Group Mission. http://www.bmwgroup.com/bmwgroup_prod/e/nav/index.html?http://www.bmwgroup.com/bmwgroup_prod/e/0_0_www_bmwgroup_com/unternehmen/unternehmensprofil/strategie/strategie.html3. Web Bike World. http://www.webbikeworld.com/Motorcycle-news/blog/4. Hoffman, Nicole P. An Examination of the † Sustainable Competitive Advantage† Concept: Past, Present, and FutureAcademy of Marketing Science Review [Online] 20005.BMW Berlin Manufacturing Planthttp://www.bmw-werk-berlin.de/berlin/htdocs/english/produktion/logistik/index_logistik.htmlThe Motorcycles:1.BMW Motorcycles. http://www.bmwmotorcycles.com/index.jsp2.Suzuki Motorcycles. http://www.suzukicycles.com/3.Kawasaki Motorcycles. http://www.kawasaki.com4.Yamaha Motorcycles. http://www.yamaha-motor.com5.Honda Motorcycles. http://powersports.honda.com/motorcycles

Juvenile Crime Boot Camps essays

Juvenile Crime Boot Camps essays Thirteen dead, over twenty wounded in a suburb of Denver Colorado, from a threat far greater than any Iranian terrorists. The threat is from within, the threat is our children. Youth Violence is a more common occurrence than the media would like us to believe. Similar instances have happened many times before: in Paducah, KY, three students were killed by a fellow student during a morning prayer circle(Leung). Also in Pearl MS, two were killed over a simple breakup (CNN), and in Springfield, OR, a sixteen-year-old killed one youth and critically wounded twenty-three over the alienation from his schoolmates (Barnard). Not to mention the sad atrocity in New Jersey where a young teen couple threw their newborn kid into a dumpster on a subfreezing night(Bad Seeds). This kind of delinquency is a problem, luckily all problems have solutions. Unfortunately the proper solutions are simply overlooked for the similar programs that address the problems cheaper. So the public believes that this type of treatment will do. However, we need something new, something radical to curb the growing violence among the youth in America. Society needs boot camps. Not just any type of boot camp. Those have been tried, and in some places, they worked, but not well enough. For any juvenile treatment to work research shows that it must address three basic needs of all children (NCCP): While many of the instituted plans have included some of the above goals they have not included all due to the cost of implementation. The problem affects kids of ages as shown in a survey taken in 1992, sixty-five percent of kids ages seven to ten feared they would die young, while that fear was shared by forty-two percent of eleven to seventeen year olds. (NCPC Report) With the arrest rate of juveniles increasing significantly during that time period, I dont blame kids for being scared. (Canada and the World) I have the solution, and recommendations for the...

French revolution Essay Example | Topics and Well Written Essays - 1250 words

French revolution - Essay Example The revolution reached its climax in 1789 upon the reconvening of the estates-general, France’s ancient legislative arm, when it became apparent that the higher class had refused to give away their privileges in the interest of saving the nation’s crippling economy. The ordinary French citizens seized this chance to force a revolution. The revolution thus got born out of a battle to attain equality and remove oppression and thus reshaped France’s social and political dimension. The French revolution served an unexpected blow to the nobles. The revolution saw the abolition of privilege and the declaration of rights of man and citizen (Hunt 62). The document of declaration made it clear that each French citizen was equal. The nobles had managed to monopolize all of the country’s wealth and had become adamant in their refusal to share the tax burden that got inflicted on the country’s wealth. With the onset of the revolution, a few nobles who sensed da nger switched sides and fought for the revolution. The nobles who still remained adamant to their privileges faced the fury of the revolution mob (Tackett 101). Many got sent to the guillotine. The revolution achieved the aim of bringing equality among the French citizens. The abolition of privilege also saw a new tax system get put in place where every citizen got to pay his tax share according to the wealth he possessed. Nobility got completely ended. The revolution also saw the abolition of church privileges that impacted on the clergy. The clergy got considered as first class citizens and most of them made up the noble class. Most of the clergy were bishops who got nominated by the king. The clergy got viewed in the same league as the aristocrats. Church property got confiscated early in the revolution. Church lands became nationalized and got sold leading to a full tenth of France’s lands to change hands. The revolution brought a massive redistribution of land that previ ously got consigned to the clergy. In present day France, church property belongs to the locals (Tackett 33). Priests got demanded to take new oaths of allegiance and loyalty to the state. Those who refused got imprisoned, executed or went into hiding. The clergy got employed as salaried officials of the state. The revolution also provided a beacon of hope and freedom to the lives of the ordinary people in France at the time. The revolution led to the creation of new political forces that centered on democracy and nationalism. This new forces gave the ordinary people hope of having an equitable and just society (Hunt 101). The revolution saw the birth of a new government system that shunned monarchy and aristocracy. The ordinary people became the significant beneficiaries of the fruits of the revolution. In the old order French society, not everyone got to enjoy rights. The revolution made a huge step towards having all men enjoy equal rights. The document of declaration of rights o f man and citizen showed promise to the ordinary people who got placed in the lower echelons of society. The French revolution did not only impact and shape the political arena in France; the revolution had a far reaching political consequence on the continent of Europe too. Most European countries at the time of the French revoluti

Murder and Manslaughter Assignment Example | Topics and Well Written Essays - 250 words

Murder and Manslaughter - Assignment Example Murders are divided into first and second degree of murder. First-degree murder is when the accused planned and premeditated about the murder. It carries the highest penalties unlike second degree of murder that was not premeditated or planned (Great Britain, 2006). Manslaughter is illegal killing without malice though it disregards human life. Murder can be reduced to manslaughter in two ways. The first one is heat of passion and the second is imperfect defense of oneself or others. In order for a killing to qualify as caused by passion, the accused must have been provoked compelling him to act irrationally with intense emotion. Lastly, the provocation should be force an average person  to act recklessly. Murder can also be reduced to manslaughter if the accused killed to defend himself or another person. In a scenario where the victim posed a threat to another person’s life, and is killed because of the danger he posed, the accused should not be guilty. However, if the victim’s threat could not have injured the other person, the murder is reduced to manslaughter. The accused charged as per the laws of the country (Yeo & University of Sydney,