[Quantitative PCR-analysis: development of a system for determining the level of an amplified DNA fragment].
A new technique is elaborated for quantitative evaluation of the polymerase chain reaction (PCR) results based on a system for yopA gene identification in Yersinia pseudotuberculosis fragment located on p45 plasmid. The analysis schedule includes amplification of the studied DNA sequence under the conditions when the reverse primer carries chemically bound biotin label on the 5'-end, hybridization of the labelled amplified fragment with the probe immobilized on the microplate surface, the probe being complementary to the inner portion of specific DNA, visualization of the label, and calculation of bound labelled DNA quantity. The lower threshold of analysis sensitivity is some picograms. It makes possible the analysis and detection of PCR product in the period of exponential increase of its content, i.e. in the initial period of reaction when the quantity of amplified DNA in the reaction mixture is not enough for reliable identification by ethidium bromide staining.
Aliapkina
Iu S
IuS
Aksenov
M Iu
MIu
Chernov
B K
BK
Gintsburg
A L
AL
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D003124:Colorimetry; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015011:Yersinia pseudotuberculosis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial
Kolichestvennyĭ PTsR-analiz: razrabotka sistemy opredeleniia soderzhaniia amplifitsirovannogo fragmenta DNK.