Abstract
Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today’s standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.
Highlights
Since the advent of enzymatic dideoxy-DNA sequencing by Frederic Sanger (Sanger et al, 1977), sequencing DNA/RNA has become standard practice in most molecular biology research
Like Sanger sequencing, today’s next-generation sequencing (NGS) technologies, with the exception of oligonucleotidebased ligation sequencing (Drmanac et al, 2010), still require a DNA polymerase to carry out the necessary biochemical reaction for replicating template sequence information
The results indicate that a single phenylalanine to tyrosine residue change (Y526) on T7 pol, homologous position (F672), of a highly conserved finger motif in A-family pols greatly reduces the enzyme’s ability to select against ddNTPs (Tabor and Richardson, 1995)
Summary
Since the advent of enzymatic dideoxy-DNA sequencing by Frederic Sanger (Sanger et al, 1977), sequencing DNA/RNA has become standard practice in most molecular biology research. The fidelity of nucleotide incorporation by X, Y, and RT Pols range from ∼10−1 to 10−4 error per base incorporated, two to three orders of magnitude lower than high-fidelity cellular DNA polymerases from A, B, or C-family enzymes (Kunkel, 2004) These repair pols generally make errors during DNA synthesis (Kunkel and Bebenek, 2000; Kunkel, 2004, 2009) and are not appropriate for high-precision DNA sequencing applications. When these enzymes incorporate an incorrect nucleotide at the primer terminus, the enzymes’ ability to extend the primer terminus diminishes, and allows the nascent DNA strand to migrate to the 3 exonuclease site for excision (See Figure 3A; Donlin et al, 1991; Joyce and Steitz, 1994; Patel and Loeb, 2001) This unique partitioning mechanism of the 3 exonuclease proofreading domain among A and B-family polymerases is disfavored for DNA sequencing. Hood’s dye-primer method simplifies traditional Sanger sequencing processes but it is not, completely ideal
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