Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Human genomic samples are complex and typically require denaturation or PCR amplification for accurate analysis.
• Directly assaying low copy number pathogen genomes or specific human DNA sequences like Single Nucleotide Polymorphisms (SNPs) without amplification presents significant challenges.

Purpose of the Study:

• To develop a novel, sensitive, and direct method for assaying genomic DNA, including pathogen and human DNA, without the need for PCR amplification.
• To demonstrate the capability of this method for detecting specific mutations (e.g., FVL G1691A, MTHFR C677T, CFTR) and low copy number targets with single base pair resolution.

Main Methods:

• Formation of a base-specific heteropolymeric triplex using native genomic DNA and an oligonucleotide third strand probe.
• Utilizing YOYO-1, a fluorescent bis-intercalator, to promote, signal, and facilitate triplex formation at room temperature in solution.
• Employing wild-type and mutant probes for specific mutation detection.

Main Results:

• The assay successfully forms specific triplex structures rapidly at room temperature without requiring a separation step.
• Homogeneous detection of genomic duplexes with single base pair resolution was achieved.
• The method demonstrated direct detection of base sequences in genomic samples, including those with human DNA, bypassing conventional PCR limitations.

Conclusions:

• A novel, sensitive diagnostic assay enables direct detection of base sequences in genomic samples, including human DNA, without PCR amplification.
• This triplex formation assay is conformation-dependent and not limited to homopurine sequences, offering a versatile alternative to traditional methods.
• The assay offers a cost-effective and efficient approach for genomic analysis, pathogen detection, and SNP genotyping.