Mutation enrichment in human DNA samples via UV-mediated cross-linking

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Detecting low-level DNA mutations is crucial for clinical diagnostics in cancer, prenatal testing, and infectious diseases.
• High abundance of wild-type (WT) alleles often masks the identification of clinically relevant genetic alterations.
• Existing methods face challenges in sensitively identifying rare mutations amidst abundant WT DNA.

Purpose of the Study:

• To introduce a novel method, UV-mediated cross-linking minor allele enrichment (UVME), for sensitive detection of low-level DNA mutations.
• To enable selective enrichment of mutated alleles over WT alleles in a single-tube PCR reaction.
• To demonstrate the utility of UVME for identifying clinically relevant genetic changes.

Main Methods:

• UVME utilizes ultraviolet (UV) irradiation (∼365 nm) to cross-link WT DNA using modified oligonucleotide probes.
• Probes with UV-sensitive nucleoside modifications bind WT sequences, facilitating UV-mediated cross-linking.
• Mismatches with mutated alleles reduce probe binding and cross-linking, favoring mutated DNA amplification during PCR.

Main Results:

• UVME successfully enriches mutated KRAS and p53 alleles from genomic DNA.
• The method allows for direct screening of enriched mutations using standard techniques like Sanger sequencing, HRM, TaqMan genotyping, or digital PCR.
• UVME enables detection of mutation allelic frequencies as low as 0.001-0.1%, depending on the detection method.

Conclusions:

• UVME is a novel and effective approach for enriching low-abundance mutated DNA alleles.
• This technique significantly improves the detection sensitivity of clinically relevant genetic mutations.
• UVME holds promise for broad applications in clinical sample analysis and molecular diagnostics.