[Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase].
The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.
Glukhov
A I
AI
Trofimova
M E
ME
Gordeev
S A
SA
Grebennikova
T V
TV
Vinogradov
S V
SV
Kiselev
V I
VI
Kramarov
V M
VM
D000255:Adenosine Triphosphate / Q000378:metabolism; D002412:Cations; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003902:Detergents; D011756:Diphosphates / Q000378:metabolism; D005784:Gene Amplification; D005814:Genes, Viral; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002412:Cations; D004279:DNA, Viral; D003902:Detergents; D011756:Diphosphates; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase
Amplifikatsiia posledovatel'nosteĭ DNK faga lambda metodom polimeraznoĭ tsepnoĭ reaktsii s ispol'zovaniem termostabil'noĭ DNK-polimerazy.