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SEQUENCING
Pages 52-70

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From page 52... ... There is much less consensus on the advisability of embarking on the determination of major amounts of its nucleotide sequence. Three kinds of reservations are often voiced: • Since the amount of useful protein-coding information in the genome is estimated to be 5 percent or less, a great deal of effort would be expended in determining the order of nucleotides of no apparent significance. Read the entire page →
From page 53... ... However, it seems fair to assume that by the time sequencing begins on a massive scale, its 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. This, of course, assumes major technological advances in sequencing, as will be described subsequently. Read the entire page →
From page 54... ... 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. In short, we anticipate that the genome sequence will serve as a basic "dictionary" that catalyzes striking advances in our understanding of cells and organisms. Read the entire page →
From page 55... ... Such spin-offs will be of great value to the biological community and are meant to augment its activities -- not to detract from them. CURRENT TECHNOLOGY IN DNA SEQUENCING: CHEMICAL AND ENZYMATIC METHODS Any project to sequence a large genome with many repeated sequences would not start with short, randomly selected genome fragments, even though this is the easiest way to obtain a large amount of sequence information quickly. Read the entire page →
From page 56... ... Thus a nested set of DNA fragments that terminate at every A nucleotide in the unknown sequence is generated. By correlating the length of the terminated chains with the identity of the base analog that was present in the reaction, one can determine the order of the nested DNA fragments and, hence, the corresponding terminal nucleotides (Figure 5-1) Read the entire page →
From page 57... ... 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 chain-terminating nucleotide (see also Figure 5-3) Read the entire page →
From page 58... ... GCAGATACGC (3') Sequence of end-labeled strand 58 Read the entire page →
From page 59... ... Both methods generate mixtures of specific DNA fragments that are separated by polyacrylamide gel electrophoresis -- a technique that can resolve fragments that differ in size by a single nucleotide. When radioactively labeled DNA fragments are used, they are detected by exposing the gel to an x-ray film. Read the entire page →
From page 61... ... DNA Fragment Label ends i Isolate endlabeled strand Cut with restriction enzyme; separate pieces Discard Expose each sample to different chemical reaction that breaks DNA after the indicated nucleotide A G C&T C eg ,-. Read the entire page →
From page 62... ... The method shown here is similar to that used in Figure 5-1. The main difference is that a fluorescently labeled primer is used to initiate the synthesis of the DNA fragments, rather than a radioactively labeled one. Read the entire page →
From page 63... ... Mixture of molecules of different lengths all terminated by A Detector 2 Mixture of molecules of different lengths all terminated by T I Mixture of molecules of different lengths all terminated by C iiiiiiiiiiinfll Mixture of molecules of different lengths all terminated by G Mixture of all DNA molecules separated by gel electrophoresis i Mill II I II 1 1 /* -- , Directic SH electro nof Dhoresis GCTA CCTGCATGGA GA CTTCGC5' 3' Smallest Largest - Chain-terminating dideoxyribonucleotides molecules molecules The molecules terminated by each dideoxyribonucleotlde are revealed as colored bands by their corresponding fluorescent primers. Read the entire page →
From page 64... ... THE ACCURACY OF DNA SEQUENCING Unless the human genome sequence is determined accurately, it will be of little use. Errors in DNA sequence determination occur at several levels. Read the entire page →
From page 65... ... This analysis puts into perspective the need to aim for approximately 0.1 percent as the maximum acceptable error rate in the initial sequence produced. EMERGING AND FUTURE TECHNOLOGY The obvious mismatch between the efficiency of current DNA-sequencing technology and the genetic complexity of genomes in even the simplest cells has given rise to several research projects aimed at developing more efficient sequencing methods. Read the entire page →
From page 66... ... This attempt to improve DNA sequencing emphasizes robotics and automated processing of samples. The automated steps begin with subcloned DNA fragments and carry them through the sequencing reactions. Read the entire page →
From page 67... ... . After the normal chemical sequencing reactions have been completed, the unlabeled samples are fractionated on a standard sequencing gel and the separated DNA fragments are transferred to a membrane while the spatial patterns of the fragments formed during electrophoresis are preserved. Read the entire page →
From page 68... ... , while allowing investigators to gain practical experience with larger scale sequencing. These improvements in current technology should be designed to reduce the cost and increase the efficiency of sequencing techniques. Read the entire page →
From page 69... ... 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 Will Require Many New Tools. Read the entire page →
From page 70... ... 1986. Automatic reading of DNA sequencing gel autoradiographs using a large format digital scanner. Read the entire page →

From page 52...

... There is much less consensus on the advisability of embarking on the determination of major amounts of its nucleotide sequence. Three kinds of reservations are often voiced: • Since the amount of useful protein-coding information in the genome is estimated to be 5 percent or less, a great deal of effort would be expended in determining the order of nucleotides of no apparent significance.

SEQUENCING Pages 52-70

SEQUENCING
Pages 52-70