Chain-terminating method, of DNA sequencing

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

Frederick Sanger, who invented the method, is among the few individuals to have won two Nobel Prizes. He received the Nobel Prize in Chemistry in 1958 for determination of the complete structure of the protein insulin and, in 1980, he received the Chemistry prize again for the discovery of the chain termination method of DNA sequencing (Chapter 14). 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

The real breakthrough was the appreciation of the limitations inherent in trying to modify RNA sequencing methods to suit the DNA problem. In this respect it is perhaps fortunate that enzymes with the type of base specificity exhibited by the RNA endonucleases are not available for degrading DNA and this also played its part in driving the development of the modern chemical and chain termination procedures for DNA sequence analysis. 

EXAMPLE 3.22 How does a dideoxynucleotide terminate the chain extension reaction in the Sanger method of DNA sequencing ... 

The chain termination, or dideoxy, method of DNA sequencing.The primer-DNA template is divided into four separate reaction mixtures, to each of which are added the four dNTPs, DNA polymerase, and one of the four ddNTPs. Synthesis is interrupted at every possible site.The mixtures of oligonucleotides are separated by polyacrylamide gel electrophoresis.The base sequence of the DNA complement is read from the bottom to the top (from the 5 end to the 3 end) of the developed gel. 

In the chain termination or dideoxy method of DNA sequencing developed by Frederick Sanger, a primer-DNA template is divided into four separate reaction mixtures. To each is added the four dNTPs, one of which is labeled... 

Fig. 4. Nucleic acid sequencing. Scheme showing the chain termination method of Sanger applied to the determination of the nucleotide sequence of a hypothetical length of DNA. a = the number shown in the column below is the number of nucleotides in the DNA chain that is complementary to the original DNA it takes no account of the constant number of nucleotides in the primer, P, attached at the 5 -end. b = nucleotide. Comp = complementary.

In the chain termination or dideoxy method of DNA sequencing developed by... 

Just after 1975, several methods for DNA sequencing were developed. Two of them, the chemical cleavage method of Allan Maxam and Walter Gilbert, and the chain terminator method of Frederick Sanger, are today used. The chemical cleavage method is explained below. 

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... 

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. 

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.

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