Rapid allelic discrimination from real-time DNA amplification.
A rapid method based on fluorescence resonance energy transfer and real-time polymerase chain reaction (PCR) is used to identify the Factor V genotype or to identify the bacterial species Bartonella qunitana or Bartonella henselae. Allelic discrimination was performed on the post-PCR product. Thermal cyclers other than the 7700 sequence detection system can be used for PCR, after which the products can be transferred to the 7700 sequence detection system for measurement of fluorescence. The Delta R (the change in fluorescence) for each dye can be collected at the final thermal cycle and an xy scatterplot used to identify the specific genotype based on graph location. There are many advantages to this method. A maximum of 96 samples can be genotyped in less than 2 h. The method tolerates a wide range of DNA concentrations and can be determined without prior DNA determination. Fluorescence is very sensitive, with a low failure rate for allelic discrimination.
Sevall
J S
JS
Specialty Laboratories Inc., 2211 Michigan Avenue, Santa Monica, California 90404, USA. SSevall@Specialtlabs.com
D000483:Alleles; D018416:Bartonella henselae / Q000235:genetics*; D018413:Bartonella quintana / Q000235:genetics*; D001483:Base Sequence; D005165:Factor V / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D005165:Factor V