Techniques Used in DNA Sequencing

The most widely used technique for determining the nucleotide sequence of DNA is the dideoxy chain termination method. Newer developments in DNA sequencing techniques make the process more automated and consequently more rapid.

Dideoxy Chain Termination Method

The fundamental element of the dideoxy chain termination method is an in vitro DNA synthesis reaction. Like any DNA synthesis reaction, a sequencing reaction requires:

■ A single-stranded piece of DNA, the template, from which a complementary copy is synthesized. ■ template, p. 168

■ A short single-stranded piece of DNA, a primer, that anneals to its complementary sequence on the single-stranded template and from which DNA synthesis can begin. Often, the DNA being sequenced has been cloned into a vector. Because the nucleotide sequences of vectors are known, a primer that anneals to a portion of the vector adjacent to the DNA to be sequenced can be readily obtained. ■ primer, p. 171

■ DNA polymerase, the enzyme that catalyzes DNA synthesis. ■ DNA polymerase, p. 171

■ Each of the four deoxynucleotides that are used in DNA synthesis—dATP, dGTP, dCTP, and dTTP; to facilitate detection of the newly synthesized DNA, one of these is labeled with a detectable marker such as a radioisotope. ■ nucleotide, p. 31

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238 Chapter 9 Biotechnology and Recombinant DNA

3'OH

3'OH

3'OH

Incorporation of a deoxynucleotide(dNTP) elongates the chain. A subsequent nucleotide can be added to the 3'OH.

Incorporation of a dideoxynucleotide (ddNTP); the ddNTP lacks a 3'OH.

Chain elongation is terminated. No additional nucleotides can be added due to the lack of a 3'OH.

Figure 9.19 Chain Termination by a Dideoxynucleotide Once a dideoxynucleotide is incorporated into a growing strand, subsequent nucleotides cannot be added due to the lack of a 3'OH.

If these were the only ingredients in the reaction, then a full-length molecule complementary to the template DNA would be synthesized. A key additional ingredient is added, however, that can terminate DNA synthesis before a full-length complement is made. This key ingredient is a dideoxynucleotide (ddNTP). Dideoxynucleotides are identical to their deoxynucleotide (dNTP) counterparts except they lack the 3'OH group, the portion of a nucleotide required for the addition of subsequent nucleotides during DNA synthesis (figure 9.19). Because they lack the 3'OH group, dideoxynucleotides are chain terminators. When one of these nucleotides is incorporated into a growing strand of DNA, no additional nucleotides can be added, and elongation of that strand ceases.

To perform a sequencing reaction, DNA polymerase, template DNA, primer, and each of the four deoxynucleotides are mixed together and then apportioned equally into four separate tubes (figure 9.20). Each tube then receives a very small amount of a different dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). In other words, ddATP is added to one

Primer

Template strand

3' Direction of chain elongation ^

' ATAGTACCTAG

Figure 9.20 Dideoxy Chain Termination Method for Determining the Nucleotide Sequence of DNA

Template strand

' ATAGTACCTAG

Dideoxynucleotide

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