Mapping and Sequencing the Human Genome (1988) / Chapter Skim
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5. Sequencing
5. Sequencing
Pages 56-74
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From page 56... ...
Since that time, with the advent of rapid techniques, about 15 million nucleotides of DNA sequence have accumulated in the GenBank database (see Chapter 6) , of which over 2 million are from human DNA (Howard Bilofsky of Bolt Beranek and Newman, Inc., personal communication, 19871.
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From page 57... ...
Given the present state of sequencing technology, this is certainly the case; for this reason, we would anticipate that most human genome sequencing in the immediate future will be carried out on cDNA clones, which represent the expressed DNA sequence. However, it seems fair to assume that by the time sequencing begins on a massive scale, the technology will have matured so far that inserting a preliminary step that discriminates among genes, intergenic regions, and introns which will presumably involve sorting out all the repeated isolates of the same DNA clones will be less efficient than sequencing large regions from ordered genomic DNA clone libraries without reference to their contents.
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From page 58... ...
However, such comparisons are rarely necessary for picking out coding sequences since existing analytical tools are adequate to identify them within a single DNA sequence. The standard procedure is to use computer programs to identify open reading frames, which are regions of nucleotide sequence lacking the stop codons that terminate a protein sequence.
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From page 59... ...
Likewise, a large group of candidate human genes will be immediately available as potential analogs of any newly discovered yeast, nematode, or Drosophila protein, for example. Other novel uses of the genome sequence data, unforeseen at present, will be developed by individual scientists, just as many of the most important current uses of recombinant-DNA technology were not foreseen by its early developers.
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From page 60... ...
Accumulating large amounts of DNA sequence data will have an impact on the biological community in other ways as well. The information contained within the genome sequence will allow full investigation into the nature and extent of polymorphism, or diversity (see Chapter 4)
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From page 61... ...
One of these, developed by Sanger and his colleagues at the Medical Research Council in Cambridge, England, is a method called enzymatic sequencing (Sanger et at., 19771. The unknown sequence is subcloned into a single-stranded DNA virus, and DNA synthesis is initiated from a primer sequence adjacent to the unknown sequence.
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From page 62... ...
THE DIFFICULTY OF DETERMINING THE SEQUENCE OF THE HUMAN GENOME WITH CURRENT TECHNOLOGY What constrains efforts to embark immediately on a large-scale human genome sequencing project? The cost and inefficiency of current DNA sequencing technologies are too great to make it feasible to contemplate determining the 3 billion nucleotides of the DNA sequence in the human genome within a reasonable time.
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From page 63... ...
The key to this method is the use of a dideoxyribonucleoside triphosphate that blocks the addition of the next nucleotide after its incorporation into the growing chain. The primed in vitro synthesis of DNA molecules in the presence of a minor proportion of a single-type of such a chain-terminating nucleotide generates a family of DNA fragments each of which ends in the particular chainterminating nucleotide (see also Figure 5-3)
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From page 64... ...
G C A G A T A C G C (3') Sequence of end-labeled strand Expose each sample to different chemical reaction that breaks C DNA after the indicated nucleotide Parallel gel electrophoresis and autoradiography FIGURE 5-2 DNA sequencing by the chemical method.
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From page 65... ...
Most of the time spent in a sequencing project is occupied with obtaining the original DNA clones containing the gene of interest and subcloning and handling the DNA prior to performing the actual sequencing reactions steps that have not yet been streamlined or automated. In addition, the entire process from subcloning to interpreting gels requires careful supervision of personnel; a ratio of no more than three technicians for each doctoral-level scientist is generally accepted as optimal.
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From page 66... ...
A careful and experienced laboratory probably achieves an accuracy of about one error in every 5,000 nucleotides (0.02 percent error rate) in the finished DNA sequence, but this degree of precision requires careful attention to virtually every nucleotide in the sequence (E.
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From page 67... ...
We seem to be on the threshold of a new generation of sequencing methods that should make large-scale sequencing projects more practical. Given the emergent state of these technologies, however, it is not surprising that expert opinion is widely divided on several key questions.
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From page 68... ...
A third approach, called multiplex sequencing, depends less on automation and more on increasing the amount of sequence data that can be obtained from one set of chemical sequencing reactions fractionated on a single sequencing gel. Each sample analyzed contains a mixture of 40 or more DNA samples, each of which has been marked with a unique short nucleotide sequence (an oligonucleotide sequence)
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From page 69... ...
In principle, if a dozen sets of 40 mixed samples each are subjected to this analysis on a single gel and each can generate 250 nucleotides of DNA sequence, then a sequence of 120,000 nucleotides can be derived from one set of chemical sequencing reactions by using sequential hybridization with the membrane produced by this method (G. Church, Harvard University, personal communication, 1987~.
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From page 70... ...
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From page 72... ...
that will be used by the biological and biomedical research community long into the future. The committee concluded that without a special effort to achieve this goal, the desired DNA sequences are not likely to be obtained in the time optimal for future medical and scientific advances, if ever.
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From page 73... ...
However, as the physical map develops and as the cost and efficiency of DNA sequencing improve, ever-larger sequencing efforts taken on by groups interested primarily in the sequence of the genome as a goal in and of itself will evolve. To Derive the Full Benefit of the Human Genome Sequence Wig Require Many New Tools, Including a Comprehensive Database of DNA Sequences from Other Organisms Comparative sequence analysis has proven a powerful technique for distinguishing those elements of a gene sequence that are highly constrained functionally from those that are not.
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From page 74... ...
1982. Codon preference and its use in identifying protein coding regions in long DNA sequences.
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