Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases.
The nucleotide-binding site in a variety of DNA polymerases was probed by analyzing incorporation of dideoxy and acyclic chain terminators. Family B archaeon DNA polymerases Vent, Deep Vent, 9 degrees N and Pfu incorporated acyclic in preference to dideoxy terminators, while the Family A DNA polymerases Taq and Klenow preferred dideoxy terminators. These divergent biases suggest that significant differences exist in sugar recognition in these two classes of polymerases. Mutants of Vent (A488L) and Taq (F667Y) that increase incorporation of dideoxy terminators maintained the acyclic/dideoxy bias of the parent enzyme, while more efficiently incorporating both dideoxy and acyclic terminators. The preference of archaeon DNA polymerases for acyclic analogs was exploited in chain terminator DNA sequence and genotype analysis. This technology was additionally aided by identification of specific dye-modified bases that improve terminator incorporation over that of the unmodified terminator. These three enhancing effects, (i) acyclic analogs, (ii) archaeon variants and (iii) specific dyes, appear to act additively and independently to increase terminator incorporation efficiency, collectively enhancing incorporation approximately 8000-fold over the wild-type incorporation of dideoxynucleotides. Fluorescent chain terminator DNA sequence traces demonstrate the applicability of these advances in improving terminator incorporation, as required in DNA sequence and genotype determinations.
Gardner
Andrew F
AF
New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Jack
William E
WE
D019943:Amino Acid Substitution / Q000235:genetics; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004396:Coloring Agents / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D015224:Dideoxynucleosides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D017186:Titrimetry
D019843:Archaeal Proteins; D004396:Coloring Agents; D015224:Dideoxynucleosides; D009711:Nucleotides; C088944:Deep Vent DNA polymerase; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; C070331:Tli polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase