A novel multiplex fluorescent competitive PCR for copy number variation detection

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Copy number variation (CNV) is a crucial genetic marker in diseases like cancer.
• Existing methods like MLPA and multiplex competitive PCR face challenges in optimization and template synthesis.
• There is a need for efficient and accurate CNV detection methods.

Purpose of the Study:

• To introduce a novel multiplex fluorescent competitive PCR (NMFC-PCR) for detecting CNVs.
• To establish a stable multiplex PCR system and obtain internal competitive templates efficiently.
• To validate the accuracy and convenience of NMFC-PCR for genetic analysis.

Main Methods:

• Developed NMFC-PCR utilizing blunt hairpin primers to reduce non-specific amplification.
• Employed limited cycles' amplification to generate internal competitive templates.
• Applied NMFC-PCR to detect CNVs in LIM homeobox 1 (LHX1) and T-box 6 (TBX6) genes in clinical samples.

Main Results:

• NMFC-PCR accurately inferred gender information from X-chromosomal to autosomal copy number ratios.
• Detected deletions in LHX1 (2 samples) and TBX6 (9 samples) in clinical specimens.
• Results were consistent with array-based comparative genomic hybridization (aCGH) and showed no CNVs in 60 normal samples.

Conclusions:

• NMFC-PCR is a cheap, efficient, accurate, and convenient method for CNV detection.
• The method simplifies the process by avoiding artificial template synthesis.
• NMFC-PCR offers a promising alternative for genetic marker analysis in various diseases.