Manual & Automated Techniques Are Available to Determine the Sequence of DNA
المؤلف:
Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil
المصدر:
Harpers Illustrated Biochemistry
الجزء والصفحة:
32nd edition.p451
2025-10-27
75
The segments of specific DNA molecules obtained by recombinant DNA technology can be analyzed to determine their nucleotide sequence. DNA sequencing depends on having a population of identical DNA molecules. This requirement can be satisfied by cloning the fragment of interest, either using the techniques described earlier, or by using PCR methods. The manual enzymatic Sanger method employs specific dideoxynucleotides that terminate DNA strand syn thesis catalyzed by DNA polymerase, at specific nucleotides as the strand is synthesized on purified single-stranded template DNA. The reactions are adjusted so that a population of DNA fragments representing termination at every nucleotide is obtained. By having a radioactive label incorporated at the termination site, one can separate the fragments according to size using high-resolution polyacrylamide gel electrophoresis. The resulting gel is vacuum dried onto filter paper and used to expose an x-ray film or more commonly a special imaging plate, which allows the visualization of the labeled DNA fragments generated during sequencing. These images are read in order to give the DNA sequence as shown in Figure 1, which is an example of first-generation, Sanger DNA sequencing. So-called next-generation sequencing (NGS), or second generation techniques are automated, but still utilized DNA polymerase and utilize four different fluorescent labels, one rep resenting each nucleotide. Each incorporated labeled deoxynucleotide emits a specific signal on excitation by a laser beam of a particular wavelength that is measured by sensitive detectors, and these signals can be recorded by a computer. The newest DNA sequencing machines use fluorescently labeled nucleotides but detect incorporation using microscopic optics. Such sequencing machines have reduced the cost of DNA sequencing by orders of magnitude, and the ensuing cost reductions, and massive parallelization of second-generation sequencing have ushered in the era of personalized genome sequencing. Third-generation DNA sequencing, though still in full development, enables real-time single-molecule sequencing of very long DNA and RNA molecules. There are currently two dis tinct third-generation technologies. One approach still uses DNA polymerase (PacBio) while the other utilizes changes in ion flow that occur when single-stranded nucleic acid molecules pass through nanometer scale nanopores (Oxford Nano pore Technologies [ONT]). These two new technologies can read the sequence of nucleic acids that range in sizes up to 100 to 900 kilobases. Such third-generation sequencers promise to yet again revolutionize the field of nucleic acid sequencing.
Fig1. Sequencing of DNA by the chain termination method devised by Sanger. The ladder-like arrays represent, from bottom to top, all of the successively longer fragments of the original DNA strand. Knowing which specific dideoxynucleotide reaction was conducted to produce each mixture of fragments, one can determine the sequence of nucleotides from the unlabeled end toward the labeled end (*) by reading up the gel. The base-pairing rules of Watson and Crick (A–T, G–C) dictate the sequence of the other (complementary) strand. (Asterisks signify site of radiolabeling.) Schematically shown (left, middle) are the terminated synthesis products of a hypothetical fragment of DNA, sequence listed (middle, top). An autoradiogram (right) of an actual set of DNA sequencing reactions that utilized the four 32P-labeled dideoxynucleotides indicated at the top of the scanned autoradiogram (dideoxy(dd)G, ddA, ddT, ddC). Electrophoresis was from top to bottom. The deduced DNA sequence is listed on the right side of the gel. Note the log-linear relationship between distance of migration (ie, top to bottom of gel) and DNA fragment length. Current state-of-the-art DNA sequencers no longer utilize gel electrophoresis for fractionation of labeled synthesis products. Moreover, in the majority of NGS sequencing platforms, synthesis is followed by monitoring incorporation of the four fluorescently labeled dXTPs.
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