Application of fluorescent dUTP on PCR and automated PCR product detection by using internal lane size standard.
A convenient, simple, cost effective and efficient fluorescent PCR labeling for automated detection was developed by using fluorescent deoxyuridine triphosphate-(F)dUTP without changing any PCR reaction parameters. Multiple fluorescent (F)dUTPs in a broad range of concentration were tested on PCR reaction and then titration tests for optimum sample loading amount with automated detection were done by using Genescan 672 software on ABI 373A DNA sequencer. PCR product were automaticaly detected although the sensitivity of each color was different. Colors of (F)dUTP and (F)dUTP/dTTP ratio showed a significant influence on the incorpration of (F)dUTP in PCR reaction, so consequently the ratio gave a great impact on the PCR product detection results. To much incorpration of (F)dUTP changed the PCR fragment mobility and also sizing value. The application of (F)dUTP should have a great potential on linkage studies, mapping with microsatellite and mutation detection.
Song
S
S
Technology Center, Perkin Elmer Corporation, Chiba-ken, Japan.
D003116:Color; D003856:Deoxyuracil Nucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D013942:Thymine Nucleotides
D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; C024157:thymidine 5'-triphosphate