Such labelling is not only important in terms of matching patients to their most appropriate drug, but also for working out what their drug dose should be and their level of risk in terms of adverse events. Individual genetic profiling is already being used routinely to prescribe therapies for patients with HIV, breast cancer, lymphoblastic leukaemia and colon cancer and in the future will be used to tailor treatments for cardiovascular disease, cancer, asthma, Alzheimer's disease and depression.
Drug developers are also using pharmacogenomic data to design drugs which can be targeted at subgroups of patients with specific genetic profiles. Although scientists established DNA had a double helix structure in , it was to be many more years before they could analyse DNA fragments.
In part this reflected the fact that small DNA molecules contain several thousands of nucleotides and it was difficult to obtain large quantities of homogeneous DNA. Scientists also lacked the means to degrade DNA which they needed for sequence analysis. A new chapter opened up in the s with the emergence of techniques to sequence ribonucleic acid RNA s. Critically, Wu's approach broke the DNA sequence down into several different components for analysis, thereby circumventing the need for large quantities of homogeneous DNA.
Subsequently, in , Wu demonstrated his method could sequence the ends of DNA in lambda phage, and two years later that it had the capacity to determine the sequence of any DNA. In Sanger, together with Alan Coulson, published what became known as the 'Plus and Minus' technique.
This enabled the sequencing of up to 80 nucleotides in one go. Three years later, in , Sanger and his colleagues announced another technique called the 'Sanger method' or 'dideoxy sequencing'. This made it possible to sequence much longer stretches of DNA very rapidly. Their approach appeared alongside the reporting of another technique by Allan Maxam and Walter Gilbert at Harvard University. While the Maxam-Gilbert method initially proved the most popular, it soon fell out of favour because it necessitated the use of hazardous chemicals and radioisotopes.
Added to this, the method it was difficult to scale-up and could not be used in standard molecular biology kits because of its technical complexity. By contrast, the Sanger method gained popularity because it was easier to use and more reliable. It was also amenable to automation, paving the way to the first generation of automated DNA sequencers.
These machines used capillary electrophoresis rather than gel electrophoresis using slabs. Several new DNA sequencing methods and machines have been developed since the s. These were built following the introduction of microfluidic separation devices which improved sample injection and speeded up separation times. Such innovations improved both the efficiency and accuracy of sequencing, allowing for high-throughput sequencing, and radically lowered the cost.
Several methods have been developed for this process. These have four key steps. In the first instance DNA is removed from the cell. This can be done either mechanically or chemically. The second phase involves breaking up the DNA and inserting its pieces into vectors, cells that indefinitely self-replicate, for cloning. In the third phase the DNA clones are placed with a dye-labelled primer a short stretch of DNA that promotes replication into a thermal cycler, a machine which automatically raises and lowers the temperature to catalyse replication.
The final phase consists of electrophoresis, whereby the DNA segments are placed in a gel and subjected to an electrical current which moves them. Originally the gel was placed on a slab, but today it is inserted into a very thin glass tube known as a capillary. When subjected to an electrical current the smaller nucleotides in the DNA move faster than the larger ones. Electrophoresis thus helps sort out the DNA fragments by their size. The different nucleotide bases in the DNA fragments are identified by their dyes which are activated when they pass through a laser beam.
All the information is fed into a computer and the DNA sequence displayed on a screen for analysis. The method developed by Sanger was pivotal to the international Human Genome Project. Data from the project provided the first means to map out the genetic mutations that underlie specific genetic diseases. It also opened up a path to more personalised medicine, enabling scientists to examine the extent to which a patient's response to a drug is determined by their genetic profile.
The genetic profile of a patient's tumour, for example, can now be used to work out what is the most effective treatment for an individual.
Data from the the Human Genome Project has also helped fuel the development of gene therapy, a type of treatment designed to replace defective genes in certain genetic disorders. In addition, it has provided a means to design drugs that can target specific genes that cause disease. Beyond medicine, DNA sequencing is now used for genetic testing for paternity and other family relationships.
It also helps identify crime suspects and victims involved in catastrophes. The technique is also vital to detecting bacteria and other organisms that may pollute air, water, soil and food. In addition the method is important to the study of the evolution of different population groups and their migratory patterns as well as determining pedigree for seed or livestock. Victor Ingram breaks the genetic code behind sickle-cell anaemia using Sanger's sequencing technique.
First comprehensive protein sequence and structure computer data published as 'Atlas of Protein Sequence and Structure'. Process called repair replication for synthesising short DNA duplexes and single-stranded DNA by polymerases is published. Nobel Prize given in recognition of discovery of restriction enzymes and their application to the problems of molecular genetics. BRCA1, a single gene on chromosome 17, shown to be responsible for many breast and ovarian cancers. Method devised to isolate methylated cytosine residues in individual DNA strands providing avenue to undertake DNA methylation genomic sequencing.
Publication of complete genome sequence of the first multicellular organism, the nematode worm Caenorhabditis elegans. Complete sequences of the genomes of the fruit fly Drosophila and the first plant, Arabidopsis, are published. Paper published demonstrating possibility of using ion channel to identify individual DNA hairpin molecules. First time four bases of DNA shown to be easily identified using engineered alpha-haemoplysin pore with a molecular adaptor. Oxford Nanopore Technology decides to focus its resources on developing nanopore sequencing for DNA sequencing.
DNA sequencing proves useful to documenting the rapid evolution of Streptococcus pneumococci in response to the application of vaccines. DNA sequencing utilised for identifying neurological disease conditions different from those given in the original diagnosis. MinION successfully used to sequence Ebola virus samples in Guinea to help combat outbreak of the disease.
High throughput nanopore sequencing device PromethION 48 launched to support population genomics for human sequencing or plant genomics. Oxford Nanopore Technology's sequencing technology chosen for a population genome genomics programme for the first time Abu Dhabi Genome Programme. Respond to or comment on this page on our feeds on Facebook , Instagram or Twitter.
Facebook Twitter Donate to WiB. DNA Sequencing Definition DNA sequencing is a method used to determine the precise order of the four nucleotide bases — adenine, guanine, cytosine and thymine - that make up a strand of DNA. He was also pivotal to the development of the dideoxy chain-termination method for sequencing DNA molecules, known as the Sanger method.
This provided a breakthrough in the sequencing of long stretches of DNA in terms of speed and accuracy and laid the foundation for the Human Genome Project. He was also instrumental in the application of genetic engineering to agricultural plants to improve their output and resistance to pests, salt and drought.
He shared the Nobel Prize in for helping to discover restriction enzymes and showing their application in molecular genetics. It was based on some work he carried out in the s. Arber indicated in that restriction enzymes could be used as a tool for cleaving DNA. The enzymes are now an important tool for genetic engineering.
In she completed the sequence of the poliovirus, the longest piece of eukaryotic DNA to be sequenced at that time. She devoted her life to understanding the Epstein-Barr virus, the cause of Burkitt's Lymphoma, a deadly form of cancer.
This he achieved with Kent Wilcox in Smith was awarded the Nobel Prize for Physiology or Medicine in for his part in the discovery of the enzyme. It was the first bacterial genome to be deciphered. Later on he helped in the genomic sequencing efforts for the fruit fly and humans at Celera Genomics. He was involved in some of the early efforts to pioneer techniques for determining base sequences in nucleic acids, known known as DNA sequencing, for which he shared the Nobel Prize for Chemistry in He was the first scientist to propose the existence of intron and exons.
In Gilbert became a proponent of the theory that the first forms of life evolved out of replicating RNA molecules. The same year he began campaigning to set up the Human Genome Project.
He was also a co-founder and the first Chief Executive Officer of Biogen, a biotechnology company originally set up to commercialise genetic engineering.
Thesis: 'On the metabolism of the amino acid lysine in the animal body'. It was the first animal to have its genome sequenced. Based on his work with the nematode, Sulston helped set up the project to sequence the human genome which he did as director of the Sanger Centre. The first draft of the human genome sequence was completed in In he shared the Nobel Prize for identifying how genes regulate the life cycle of cells through apoptosis.
His work is initially supported by a Beit Memorial Fellowship from and then by Medical Research Council from In Venter worked with a team to create the first form of synthetic life. This involved synthesising a long molecule of DNA that contained an entire bacerum genome and then inserting this into another cell. The technique Sanger develops for sequencing insulin later becomes known as the degradation or DNP method.
It was the result of a collective effort led by Margaret Dayhoff to co-ordinate the ever-growing amount of information about protein sequences and their biochemical function. It provided the model for GenBank and many other molecular databases. Arber, 'Host-controlled modification of bacteriophage', Annual Review Microbiology, 19 , They found that bacteria protect themselves against invading viruses by producing two types of enzymes.
One cut up the DNA of the virus and the other restricted its growth. Arber believed these two enzymes could provide an important tool for cutting and pasting DNA, the method now used in genetic engineering.
Taq is later important in the PCR technique. Restriction enzymes are now workhorses of molecular biology. They are essential in the development of recombinant DNA and were pivotal to the foundation of the biotechnology industry.
The method provides an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene being synthesised.
Represents radical new approach which allows direct visual scanning of a sequence. It is the first DNA based organism to have its complete genome sequenced.
Sanger and his team use the plus and minus technique to determine the sequence. While still using polyacrylamide gels to resolve DNA fragments, the Maxam and Gilbert technique differed significantly in its approach.
Instead of relying on DNA polymerase to generate fragments, radiolabelled DNA is treated with chemicals which break the chain at specific bases; after running on a polyacrylamide gel the length of cleaved fragments and thus position of specific nucleotides can be determined and therefore sequence inferred see Fig.
First-generation DNA sequencing technologies. Different chemical treatments are then used to selectively remove the base from a small proportion of DNA sites. Hydrazine removes bases from pyrimidines cytosine and thymine , while hydrazine in the presence of high salt concentrations can only remove those from cytosine. Acid can then be used to remove the bases from purines adenine and guanine , with dimethyl sulfate being used to attack guanines although adenine will also be affected to a much lesser extent.
Piperidine is then used to cleave the phophodiester backbone at the abasic site, yielding fragments of variable length. The chain-termination technique makes use of chemical analogues of the deoxyribonucleotides dNTPs that are the monomers of DNA strands.
Mixing radiolabelled ddNTPs into a DNA extension reaction at a fraction of the concentration of standard dNTPs results in DNA strands of each possible length being produced, as the dideoxy nucleotides get randomly incorporated as the strand extends, halting further progression. By performing four parallel reactions containing each individual ddNTP base and running the results on four lanes of a polyacrylamide gel, one is able to use autoradiography to infer what the nucleotide sequence in the original template was, as there will a radioactive band in the corresponding lane at that position of the gel see Fig.
While working on the same principle as other techniques that of producing all possible incremental length sequences and labelling the ultimate nucleotide , the accuracy, robustness and ease of use led to the dideoxy chain-termination method — or simply, Sanger sequencing — to become the most common technology used to sequence DNA for years to come.
A number of improvements were made to Sanger sequencing in the following years, which primarily involved the replacement of phospho- or tritrium-radiolabelling with fluorometric based detection allowing the reaction to occur in one vessel instead of four and improved detection through capillary based electrophoresis.
Both of these improvements contributed to the development of increasingly automated DNA sequencing machines [27] , [28] , [29] , [30] , [31] , [32] , [33] , and subsequently the first crop of commercial DNA sequencing machines [34] which were used to sequence the genomes of increasingly complex species.
The development of techniques such as polymerase chain reaction PCR [37] , [38] and recombinant DNA technologies [39] , [40] further aided the genomics revolution by providing means of generating the high concentrations of pure DNA species required for sequencing. Improvements in sequencing also occurred by less direct routes. However, more sequenced genomes and tools for genetic manipulation provided the resources to find polymerases that were better at accommodating the additional chemical moeities of the increasingly modified dNTPs used for sequencing [42].
Eventually, newer dideoxy sequencers — such as the ABI PRISM range developed from Leroy Hood's research, produced by Applied Biosystems [43] , which allowed simultaneous sequencing of hundreds of samples [44] — came to be used in the Human Genome Project, helping to produce the first draft of that mammoth undertaking years ahead of schedule [45] , [46].
Concurrent with the development of large-scale dideoxy sequencing efforts, another technique appeared that set the stage for the first wave in the next generation of DNA sequencers. This method markedly differed from existing methods in that it did not infer nucleotide identity through using radio- or fluorescently-labelled dNTPs or oligonucleotides before visualising with electrophoresis. Instead researchers utilized a recently discovered luminescent method for measuring pyrophosphate synthesis: this consisted of a two-enzyme process in which ATP sulfurylase is used to convert pyrophosphate into ATP, which is then used as the substrate for luciferase, thus producing light proportional to the amount of pyrophosphate [47].
This approach was used to infer sequence by measuring pyrophosphate production as each nucleotide is washed through the system in turn over the template DNA affixed to a solid phase [48]. Later improvements included attaching the DNA to paramagnetic beads, and enzymatically degrading unincorporated dNTPs to remove the need for lengthy washing steps. The major difficulty posed by this technique is finding out how many of the same nucleotide there are in a row at a given position: the intensity of light released corresponds to the length of the homopolymer, but noise produced a non-linear readout above four or five identical nucleotides [51].
The sequencing machines produced by later purchased by Roche were a paradigm shift in that they allowed the mass parallelisation of sequencing reactions, greatly increasing the amount of DNA that can be sequenced in any one run [52].
Libraries of DNA molecules are first attached to beads via adapter sequences, which then undergo a water-in-oil emulsion PCR emPCR [53] to coat each bead in a clonal DNA population, where ideally on average one DNA molecule ends up on one bead, which amplifies in its own droplet in the emulsion see Fig.
These DNA-coated beads are then washed over a picoliter reaction plate that fits one bead per well; pyrosequencing then occurs as smaller bead-linked enzymes and dNTPs are washed over the plate, and pyrophosphate release is measured using a charged couple device CCD sensor beneath the wells.
This set up was capable of producing reads around — base pairs bp long, for the million or so wells that would be expected to contain suitably clonally-coated beads [52].
This parallelisation increased the yield of sequencing efforts by orders of magnitudes, for instance allowing researchers to completely sequence a single human's genome — that belonging to DNA structure pioneer, James Watson — far quicker and cheaper than a similar effort by DNA-sequencing entrepreneur Craig Venter's team using Sanger sequencing the preceding year [54] , [55]. This principle of performing huge numbers of parallel sequencing reactions on a micrometer scale — often made possible as a result of improvements in microfabrication and high-resolution imaging — is what came to define the second-generation of DNA sequencing [57].
Second-generation DNA sequencing parallelized amplification. Bead-DNA conjugates can then be emulsified using aqueous amplification reagents in oil, ideally producing emulsion droplets containing only one bead illustrated in the two leftmost droplets, with different molecules indicated in different colours. Clonal amplification then occurs during the emPCR as each template DNA is physically separate from all others, with daughter molecules remaining bound to the microbeads.
This is the conceptual basis underlying sequencing in , Ion Torrent and polony sequencing protocols. Single-stranded DNA with terminating sequences complementary to the two lawn-oligos will anneal when washed over the flow-cell, and during isothermal PCR will replicate in a confined area, bending over to prime at neighbouring sites, producing a local cluster of identical molecules. DNA polymerase can then be added to the wells, and each nucleotide can be washed over in turn, and dNTP incorporation monitored e.
Flow-cell bound clusters produced via bridge amplification d can be visualized by detecting fluorescent reversible-terminator nucleotides at the ends of a proceeding extension reaction, requiring cycle-by-cycle measurements and removal of terminators. A number of parallel sequencing techniques sprung up following the success of The most important among them is arguably the Solexa method of sequencing, which was later acquired by Illumina [56].
Instead of parallelising by performing bead-based emPCR, adapter-bracketed DNA molecules are passed over a lawn of complementary oligonucleotides bound to a flowcell; a subsequent solid phase PCR produces neighbouring clusters of clonal populations from each of the individual original flow-cell binding DNA strands [58] , [59]. These modified dNTPs and DNA polymerase are washed over the primed, single-stranded flow-cell bound clusters in cycles.
At each cycle, the identity of the incorporating nucleotide can be monitored with a CCD by exciting the fluorophores with appropriate lasers, before enzymatic removal of the blocking fluorescent moieties and continuation to the next position. While the first Genome Analyzer GA machines were initially only capable of producing very short reads up to 35 bp long they had an advantage in that they could produce paired-end PE data, in which the sequence at both ends of each DNA cluster is recorded.
This is achieved by first obtaining one SBS read from the single-stranded flow-cell bound DNA, before performing a single round of solid-phase DNA extension from remaining flow-cell bound oligonucleotides and removing the already-sequenced strand. Having thus reversed the orientation of the DNA strands relative to the flow-cell, a second read is then obtained from the opposite end of the molecules to the first.
As the input molecules are of an approximate known length, having PE data provides a greater amount of information. This improves the accuracy when mapping reads to reference sequences, especially across repetitive sequences, and aids in detection of spliced exons and rearranged DNA or fused genes.
The standard Genome Analyzer version GAIIx was later followed by the HiSeq, a machine capable of even greater read length and depth, and then the MiSeq, which was a lower-throughput but lower cost machine with faster turnaround and longer read lengths [61] , [62]. A number of other sequencing companies, each hosting their own novel methodologies, have also appeared and disappeared and had variable impacts upon both what experiments are feasible and the market at large.
As its name suggests, SOLiD sequenced not by synthesis i. While the SOLiD platform is not able to produce the read length and depth of Illumina machines [66] , making assembly more challenging, it has remained competitive on a cost per base basis [67].
The last remarkable second-generation sequencing platform is that developed by Jonathan Rothburg after leaving This technology allows for very rapid sequencing during the actual detection phase [67] , although as with and all other pyrosequencing technologies it is less able to readily interpret homopolymer sequences due to the loss of signal as multiple matching dNTPs incorporate [70]. The capabilities of DNA sequencers have grown at a rate even faster than that seen in the computing revolution described by Moore's law: the complexity of microchips measured by number of transistors per unit cost doubles approximately every two years, while sequencing capabilities between and doubled every five months [71].
The various offshoot technologies are diverse in their chemistries, capabilities and specifications, providing researchers with a diverse toolbox with which to design experiments. However in recent years the Illumina sequencing platform has been the most successful, to the point of near monopoly [72] and thus can probably considered to have made the greatest contribution to the second-generation of DNA sequencers.
There is considerable discussion about what defines the different generations of DNA sequencing technology, particularly regarding the division from second to third [73] , [74] , [75] , [76].
Arguments are made that single molecule sequencing SMS , real-time sequencing, and simple divergence from previous technologies should be the defining characteristics of the third-generation.
It is also feasible that a particular technology might straddle the boundary. Here we consider third generation technologies to be those capable of sequencing single molecules, negating the requirement for DNA amplification shared by all previous technologies. While relatively slow and expensive and producing relatively short reads , this was the first technology to allow sequencing of non-amplified DNA, thus avoiding all associated biases and errors [73] , [75].
As Helicos filed for bankruptcy early in [80] other companies took up the third-generation baton. At the time of writing, the most widely used third-generation technology is probably the single molecule real time SMRT platform from Pacific Biosciences [81] , available on the PacBio range of machines. During SMRT runs DNA polymerisation occurs in arrays of microfabricated nanostructures called zero-mode waveguides ZMWs , which are essentially tiny holes in a metallic film covering a chip.
These ZMWs exploit the properties of light passing through apertures of a diameter smaller than its wavelength, which causes it to decay exponentially, exclusively illuminating the very bottom of the wells. This allows visualisation of single fluorophore molecules close to the bottom of the ZMW, due to the zone of laser excitation being so small, even over the background of neighbouring molecules in solution [82]. This process can sequence single molecules in a very short amount of time.
The PacBio range possesses a number of other advantageous features that are not widely shared among other commercially available machines. As sequencing occurs at the rate of the polymerase it produces kinetic data, allowing for detection of modified bases [84]. PacBio machines are also capable of producing incredibly long reads, up to and exceeding 10 kb in length, which are useful for de novo genome assemblies [73] , [81].
Third-generation DNA sequencing nucleotide detection. As the diameter is narrower than the excitation light's wavelength, illumination rapidly decays travelling up the ZMW: nucleotides being incorporated during polymerisation at the base of the ZMW provide real-time bursts of fluorescent signal, without undue interference from other labelled dNTPs in solution.
As the ssDNA passes through the nanopore the different bases prevent ionic flow in a distinctive manner, allowing the sequence of the molecule to be inferred by monitoring the current at each channel. Perhaps the most anticipated area for third-generation DNA sequencing development is the promise of nanopore sequencing, itself an offshoot of a larger field of using nanopores for the detection and quantification of all manner of biological and chemical molecules [85].
Moreover, passage through the channel blocks ion flow, decreasing the current for a length of time proportional to the length of the nucleic acid [86]. There is also the potential to use non-biological, solid-state technology to generate suitable nanopores, which might also provide the ability to sequence double stranded DNA molecules [87] , [88]. Despite the admittedly poor quality profiles currently observed, it is hoped that such sequencers represent a genuinely disruptive technology in the DNA sequencing field, producing incredibly long read non-amplified sequence data far cheaper and faster than was previously possible [92] , [90] , [85].
Already MinIONs have been used on their own to generate bacterial genome reference sequences [93] , [94] and targeted amplicons [95] , [96] , or used to generate a scaffold to map Illumina reads to [97] , [98] , [96] , combining the ultra long read length of the nanopore technology and the high read depth and accuracy afforded by the short read sequencing. The fast run times and compact nature of the MinION machine also presents the opportunity to decentralize sequencing, in a move away from the core services that are common today.
They can even be deployed it in the field, as proved by Joshua Quick and Nicholas Loman earlier this year when they sequenced Ebola viruses in Guinea two days after sample collection [99]. Nanopore sequencers could therefore revolutionize not just the composition of the data that can be produced, but where and when it can be produced, and by whom.
It is hard to overstate the importance of DNA sequencing to biological research; at the most fundamental level it is how we measure one of the major properties by which terrestrial life forms can be defined and differentiated from each other. Therefore over the last half century many researchers from around the globe have invested a great deal of time and resources to developing and improving the technologies that underpin DNA sequencing.
At the genesis of this field, working primarily from accessible RNA targets, researchers would spend years laboriously producing sequences that might number from a dozen to a hundred nucleotides in length.
Over the years, innovations in sequencing protocols, molecular biology and automation increased the technological capabilities of sequencing while decreasing the cost, allowing the reading of DNA hundreds of basepairs in length, massively parallelized to produce gigabases of data in one run. Researchers moved from the lab to the computer, from pouring over gels to running code. Genomes were decoded, papers published, companies started — and often later dissolved — with repositories of DNA sequence data growing all the while.
Therefore DNA sequencing — in many respects a relatively recent and forward-focussed research discipline — has a rich history. An understanding of this history can provide appreciation of current methodologies and provide new insights for future ones, as lessons learnt in the previous generation inform the progress of the next. National Center for Biotechnology Information , U.
Sponsored Document from. James M. Author information Article notes Copyright and License information Disclaimer. Heather: ku. This article has been cited by other articles in PMC. Abstract Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications.
First-generation DNA sequencing Watson and Crick famously solved the three-dimensional structure of DNA in , working from crystallographic data produced by Rosalind Franklin and Maurice Wilkins [2] , [3] , which contributed to a conceptual framework for both DNA replication and encoding proteins in nucleic acids.
Open in a separate window. Second-generation DNA sequencing Concurrent with the development of large-scale dideoxy sequencing efforts, another technique appeared that set the stage for the first wave in the next generation of DNA sequencers. Third-generation DNA sequencing There is considerable discussion about what defines the different generations of DNA sequencing technology, particularly regarding the division from second to third [73] , [74] , [75] , [76]. Conclusions It is hard to overstate the importance of DNA sequencing to biological research; at the most fundamental level it is how we measure one of the major properties by which terrestrial life forms can be defined and differentiated from each other.
Aa Aa Aa. How do researchers "read" gene sequences? Determining the order of the nucleotides within a gene is known as DNA sequencing. The earliest DNA sequencing methods were time consuming, but a major breakthrough came in with the development of the process called Sanger sequencing. Sanger sequencing is named after English biochemist Frederick Sanger, and it is sometimes also referred to as chain-termination sequencing or dideoxy sequencing.
Some 25 years after its creation, the Sanger method was used to sequence the human genome, and, with the addition of many technological improvements and modifications, it remains an important method in laboratories across the world today. How does Sanger sequencing work? Understanding DNA replication. Setting up the sequencing experiment. Adding ddNTPs. Figure 2: The four ddNTPs.
Figure 3: By adding together information about all of the truncated strands, researchers can determine the nucleotide sequence of the DNA target. The sugar-phosphate backbone is depicted as gray, horizontal cylinders stacked end-to-end.
Each cylinder is attached to a thin rectangle, representing the nucleotide. Gray nucleotides have an unknown chemical composition. Green nucleotides represent adenine, and orange nucleotides represent cytosine. The sequence of nucleotides is: two gray, green, orange, gray, orange, two gray, green, 5 gray, green, gray. In the bottom DNA strand, eight nucleotides are base paired with the upper strand on the right side.
The second sugar-phosphate group is colored black instead of gray, indicating that it contains a dideoxy-ribose sugar, and the first nucleotide is off-set to indicate that it is not bound to the DNA chain. The sequence of the paired nucleotides is: red thymine , blue guanine , orange, blue, green, orange, red, blue. In a smaller diagram to the left of the larger chain, examples of resulting truncated nucleotide chains help decipher the DNA sequence.
Under the heading ddTTP, three nucleotide chains are shown. The first chain contains 14 nucleotides, with a red ddTTP inserted in the left-most position, truncating synthesis. The second chain contains 8 nucleotides, also truncated with a ddTTP. The third chain contains only 2 nucleotides, truncated after ddTTP addition.
Under the heading ddGTP, two nucleotide chains are shown. The first chain contains 13 nucleotides, truncated after ddGTP addition. The second chain contains 11 nucleotides, also truncated after ddGTP addition. After complete analysis with all four ddNTPs, the final nucleotide sequence is shown in the right panel.
Nucleotides are represented by different colored rectangles: red for thymine, blue for guanine, green for adenine, and orange for cytosine. Below the sequenced strand, examples of truncated strands from the four reactions are shown. Reading the sequence: Now and then.
How is DNA sequencing used by scientists?
0コメント