A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer.

Autor:	Kimoto, Michiko, Yokoyama, Shigeyuki, Hirao, Ichiro
Předmět:	NUCLEOTIDES NUCLEIC acids DNA POLYMERASE chain reaction GELATION GENES
Zdroj:	Biotechnology Letters; Jun2004, Vol. 26 Issue 12, p999-1005, 7p
Abstrakt:	A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5′-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication. [ABSTRACT FROM AUTHOR]
Databáze:	Complementary Index
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