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Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076-80.

Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.

Author information

1
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115.

Abstract

Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis. Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate. Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage. A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis. The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.

PMID:
2657738
PMCID:
PMC287391
DOI:
10.1073/pnas.86.11.4076
[Indexed for MEDLINE]
Free PMC Article

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