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Detection of simultaneous multi-mutations using base-quenched probe.

Huihui Mao1, Guanghua Luo1, Jun Zhang1

  • 1Comprehensive Laboratory, Changzhou Key Lab of Individualized Diagnosis and Treatment Associated with High Technology Research, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China.

Analytical Biochemistry
|December 14, 2017
PubMed
Summary
This summary is machine-generated.

This study demonstrates a base-quenched probe method using real-time PCR for accurate single nucleotide polymorphism (SNP) detection. Various fluorophores successfully identified multiple mutations simultaneously, confirmed by sequencing.

Keywords:
Melting-curve analysisSingle nucleotide polymorphismThe base-quenched probe

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Single nucleotide polymorphisms (SNPs) are key genetic variations.
  • Real-time PCR with melting-curve analysis is a common SNP detection method.
  • Fluorophore-labeled probes are essential for real-time PCR assays.

Purpose of the Study:

  • To evaluate the utility of common commercial fluorophores for SNP detection using a base-quenched probe method.
  • To assess the simultaneous detection of multiple SNPs.
  • To confirm the accuracy of the method through direct sequencing.

Main Methods:

  • Application of base-quenched probes labeled with FAM, HEX, CY5, CY3, TET, JOE, Texas Red, and ROX.
  • Utilizing real-time PCR and melting-curve analysis.
  • Simultaneous detection of SNPs across different fluorescence channels.
  • Validation of results via direct sequencing.

Main Results:

  • All tested fluorophores (FAM, HEX, CY5, CY3, TET, JOE, Texas Red, ROX) showed base-dependent fluorescence, confirming their applicability for SNP detection.
  • The method successfully detected multiple mutations simultaneously.
  • The accuracy of the fluorophore-based SNP detection was validated by direct sequencing.
  • Demonstrated successful simultaneous detection of APOM rs707921, APOM rs707922, and MCP-1 rs1024611 SNPs.

Conclusions:

  • The base-quenched probe method is effective for simultaneous SNP detection using various commercial fluorophores.
  • This approach offers accurate and reliable SNP genotyping.
  • The method is suitable for multiplex SNP analysis in genetic research and diagnostics.