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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Conventional real-time PCR for allele ratio quantification in pooled DNA relies on Ct values and amplification efficiency.
• Exponential calculations in Ct methods amplify errors, leading to significant result variations.
• A new PCR data analysis strategy based on normalized fluorescence ratio has been developed.

Purpose of the Study:

• To develop and validate a novel method for accurate allele ratio quantification in pooled DNA.
• To improve the reliability, precision, and repeatability of allele ratio measurements.
• To explore the potential for fully automatic allelic ratio quantification.

Main Methods:

• Determined initial reaction background fluorescence using a four-parametric sigmoid function fit.
• Normalized fluorescence by subtracting background and dividing by specific background fluorescence.
• Generated a standard linear regression equation relating normalized fluorescence ratio to known allele ratios for extrapolation.

Main Results:

• The proposed normalized fluorescence ratio method was compared against baseline subtracted fluorescence ratio and conventional Ct methods.
• Results demonstrated improved reliability, precision, and repeatability in allele ratio quantification.
• The method shows potential for fully automatic allelic ratio determination.

Conclusions:

• The novel normalized fluorescence ratio method offers superior performance for allele ratio quantification.
• This approach enhances the accuracy and consistency of genetic analysis in pooled DNA samples.
• The method paves the way for automated and more dependable genetic analyses.