Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR.

ABSTRACT:

Background

Conventional real-time PCR to quantify the allele ratio in pooled DNA mainly depends on PCR amplification efficiency determination and Ct value, which is defined as the PCR cycle number at which the fluorescence emission exceeds the fixed threshold. Because of the nature of exponential calculation, slight errors are multiplied and the variations of the results seem too large. We have developed a new PCR data point analysis strategy for allele ratio quantification based on normalized fluorescence ratio.

Results

In our method, initial reaction background fluorescence was determined based upon fitting of raw fluorescence data to four-parametric sigmoid function. After that, each fluorescence data point was first subtracted by respective background fluorescence and th

SUBMITTER: Yu A 

PROVIDER: S-EPMC1552069 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature