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Multiplex allele-specific amplification from whole blood for detecting multiple polymorphisms simultaneously.

Jianjie Zhu¹, Lanxin Chen, Yong Mao

¹Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University, Suzhou, China.

Genetic Testing and Molecular Biomarkers

|October 18, 2012

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Directly amplifying DNA from whole blood simplifies single-nucleotide polymorphism (SNP) genotyping. This new method for polymerase chain reaction (PCR) is accurate, cost-effective, and aids in predicting gastric cancer risk.

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Allele-specific amplification via polymerase chain reaction (PCR) is standard for single-nucleotide polymorphism (SNP) genotyping.
Current methods necessitate tedious genomic DNA extraction from whole blood, increasing contamination risk.

Purpose of the Study:

To develop a direct PCR amplification method from whole blood, eliminating the need for DNA extraction.
To optimize PCR conditions for direct amplification and establish multiplex assays for SNP genotyping.

Main Methods:

Optimized PCR solution pH, magnesium ion concentration, and glycerol concentration.
Developed triplex, five-plex, and eight-plex allele-specific amplifications directly from whole blood.
Applied the method to a case-control study involving 97 gastric cancer patients and 141 controls, analyzing 14 polymorphisms.

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Main Results:

Successfully established multiplex allele-specific amplifications directly from whole blood.
Identified significant associations between SNPs rs9344, rs1799931, and rs1800629 and gastric cancer risk.
Demonstrated the method's accuracy, time-efficiency, cost-effectiveness, and ease of use.

Conclusions:

Direct PCR amplification from whole blood is a viable and advantageous alternative to traditional DNA extraction methods.
This technique facilitates high-throughput SNP genotyping and holds potential for clinical prediction of disease susceptibility, such as gastric cancer.
The optimized multiplex assays provide a robust tool for genetic association studies.