Chain-terminating method

The result of sequence determination of an oligonucleotide as performed by the Sanger dideoxy chain termination method is displayed at right. 

This method was developed by Sanger and his colleagues and is also referred to as the chain termination method. The procedure requires a single-stranded DNA template and a short primer complementary to the 3 end of the region of DNA to be sequenced. A complementary copy of the DNA strand is produced... 

Figure 13.20 The dideoxy (chain termination) method of DNA sequencing. A...

The Maxam-Gilbert method doesn t require synthesis of a primer and it sometimes works well for sequences that are difficult to obtain with the Sanger -Coulson procedure. The two methods may both be used to provide additional certainty about a sequence. The Maxam-Gilbert method is very convenient for sequencing small oligonucleotides which often react poorly with the polymerase used for the chain termination method. The method usually requires that a restriction map be prepared. 

Automated DNA sequencing uses the chain termination method but with an oligonucleotide primer labeled with a fluorescent dye. Each of the four reactions receives a primer labeled with a different dye. After incubation, the reaction mixtures are pooled and electrophoresed on one lane of a polyacrylamide gel. The order in which the different fluorescently labeled termination products elute from the gel gives the DNA sequence. 

The chain-terminator method relies on the use of the enzyme DNA polymerase I, which catalyzes the synthesis of DNA. This enzyme makes complementary copies of single-stranded DNA, starting from the 5 end and proceeding to the 3 end. First the DNA to be sequenced, termed the template strand, is isolated. To begin the synthesis, the enzyme requires the presence of a small piece of DNA, called a primer, at the 5 end of the chain to be synthesized. Because the template DNA is prepared by a restriction endonuclease, a few bases at its 3 end are known. A primer that is complementary to this sequence is prepared by chemical synthesis. The enzyme then adds the bases that are complementary to the template DNA to the 3 -hydroxy group of the primer. 

Show the pattern of the gel electrophoresis spots that would be obtained from sequencing the DNA fragment CTTAGTTGCACCT using the chain-terminator method. The primer is GA. 

An automatic sequencing instrument has been developed that uses the chain-terminator method. To avoid the use of radioactive labels, a different color fluorescent dye is attached to the primer in each of the four reactions used to synthesize the DNA fragments. The mixture of fragments from all four reactions is then analyzed using electrophoresis in a single lane. A fluorescent spot appears for each polynucleotide of increasing size. The 3 -terminal base for each spot can be determined by the color of the fluorescence. The detection system is computer controlled, and the acquisition of data is automated. A schematic representation... 

Understand the chain-terminator method for determining the sequence of DNA. 

The use of Exonuclease III for preparing single-stranded primers in connection with the chain-termination method has been considered earlier (see 3.1.3.). Exonuclease III can also be used to prepare single-stranded templates from a linear duplex DNA and Smith (1979) pioneered this approach and developed it into a fairly general method for sequencing restriction fragments. 

Poncz, M., D. Solowiejczyk, M. Baliantine, E. Schwartz and S. Surrey, 1982. Nonrandom DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc. Natl. Acad. Sci. USA 79, 4298-4302. 

Figure 6.4. Strategy of the Chain-Termination Method for Sequencing DNA. Fragments are produced by adding the 2, 3 -dideoxy analog of a dNTP to each of four polymerization mixtures. For example, the addition of the dideoxy analog of dATP (shown in red) results in fragments ending in A. The dideoxy analog cannot be extended.

Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method... 

DNA fragments up to about 500 nucleotides long are most commonly sequenced in automated instruments based on the Sanger (dideoxy chain termination) method (see Figure 9-23). 

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