Two-temperature LATE-PCR endpoint genotyping |
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Authors: | J Aquiles Sanchez Jessica D Abramowitz Jesse J Salk Arthur H Reis Jr John E Rice Kenneth E Pierce Lawrence J Wangh |
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Institution: | (1) Department of Biology, MS008, Brandeis University, Waltham, MA 02454, USA;(2) Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978, Israel;(3) Department of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98019, USA |
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Abstract: | Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau
and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal
intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method
corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous
genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to
those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances
the multiplex detection capacity of the assay. |
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