Box 6.1DNA polymerases for chain termination sequencing

Any template-dependent DNA polymerase is capable of extending a primer that has been annealed to a single-stranded DNA molecule, but not all polymerases do this in a way that is useful for DNA sequencing. Three criteria in particular must be fulfilled by a sequencing enzyme:

  • High processivity. This refers to the length of polynucleotide that is synthesized before the polymerase terminates through natural causes. A sequencing polymerase must have high processivity so that it does not dissociate from the template before incorporating a chain-terminating nucleotide.
  • Negligible or zero 5′→3exonuclease activity. Most DNA polymerases also have exonuclease activities, meaning that they can degrade DNA polynucleotides as well as synthesize them (Section 4.1.1; see Figure 4.7). A 5′→3′ exonuclease activity enables the polymerase to remove a DNA strand that is already attached to the template. This is a disadvantage in DNA sequencing because removal of nucleotides from the 5′ ends of the newly synthesized strands alters the lengths of these strands, making it impossible to read the sequence from the banding pattern in the polyacrylamide gel.
  • Negligible or zero 3′→5exonuclease activity is also desirable so that the polymerase does not remove the chain termination nucleotide once it has been incorporated.

These are stringent requirements and are not entirely met by any naturally occurring DNA polymerase. Instead, artificially modified enzymes are generally used. The first of these to be developed was the Klenow polymerase, which is a version of Escherichia coli DNA polymerase I from which the 5′→3′ exonuclease activity of the standard enzyme has been removed, either by cleaving away the relevant part of the protein or by genetic engineering (Section 4.1.1). The Klenow polymerase has relatively low processivity, limiting the length of sequence that can be obtained from a single experiment to about 250 bp, and giving non-specific bands on the sequencing gel, these ‘shadow’ bands representing strands that have terminated naturally rather than by incorporation of a ddNTP. The Klenow enzyme was therefore superseded by a modified version of the DNA polymerase encoded by bacteriophage T7, this enzyme going under the tradename ‘Sequenase’. Sequenase has high processivity and no exonuclease activity, and also possesses other desirable features such as rapid reaction rate and the ability to use many modified nucleotides as substrates.

Image ch4f7

From: Chapter 6, Sequencing Genomes

Cover of Genomes
Genomes. 2nd edition.
Brown TA.
Oxford: Wiley-Liss; 2002.
Copyright © 2002, Garland Science.

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