The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E. coli

G A Nevinsky; A V Nemudraya; A S Levina; V V Khomov

doi:10.1016/0014-5793(89)81642-4

The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E. coli

FEBS Lett. 1989 Nov 20;258(1):166-70. doi: 10.1016/0014-5793(89)81642-4.

Authors

G A Nevinsky¹, A V Nemudraya, A S Levina, V V Khomov

Affiliation

¹ Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the USSR Academy of Sciences, Novosibirsk.

PMID: 2480250
DOI: 10.1016/0014-5793(89)81642-4

Abstract

DNA synthesis at primers d(pT)n, d(pA)n, d(pC)n, and d(pG)n in the presence of corresponding complementary templates and at hetero-oligoprimers complementary to M13 phage DNA was investigated. The values of both -log Km and log Vmax increased linearly if homo-oligoprimers contained less than 10 nucleotides. The lengthening of d(pT)n and d(pA)n primers by one mononucleotide unit (n = 1-10) resulted in the 1.82-fold decrease of the Km values. The incremental decreases of Km for d(pC)n and d(pG)n were equal to about 2.46. The enhancement of the homo- and hetero-oligonucleotide primers' affinity to the enzyme due to one Watson-Crick hydrogen bond between complementary template and primer is about 1.35 times. This allows to calculate the Km values for primers of various structure and length up to 10 units. The objective laws of the Km and Vmax values changes for primers containing more than 10 nucleotides were analyzed.

MeSH terms

Algorithms
DNA Polymerase I / analysis*
DNA-Directed DNA Polymerase / analysis*
Escherichia coli / enzymology*
Kinetics
Peptide Fragments / analysis
RNA / analysis*

Substances

Peptide Fragments
RNA primers
RNA
DNA Polymerase I
DNA-Directed DNA Polymerase