Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Haplotype phasing of single nucleotide polymorphisms (SNPs) is crucial for determining disease risk and severity.
• Conventional methods struggle to phase SNPs located far apart due to DNA secondary structures and probe hybridization challenges.
• Enzyme-assisted phasing is limited by complex nucleic acid structures at room temperature.

Purpose of the Study:

• To develop a molecular method for revealing the relative positions of SNPs located 1.4 kb apart in two gene copies.
• To overcome limitations of existing methods in phasing distant SNPs and complex DNA structures.
• To enable accurate and rapid haplotype phasing for improved genetic analysis.

Main Methods:

• A competitive toehold probes and sink strategy was employed at elevated temperatures.
• The method was designed to reveal relative SNP positions in long DNA targets (1.4 kb apart).
• No additional amplification steps were required.

Main Results:

• Successfully differentiated 20 nM of 10 possible diplotypes in a long DNA target with two SNP sites 1.4 kb apart.
• Achieved accurate haplotype phasing within one hour.
• Demonstrated the method's efficacy without amplification.

Conclusions:

• The developed molecular method offers a promising approach for accurate and fast haplotype phasing of distant SNPs.
• This technology can significantly advance genetic studies and disease risk assessment.
• The method overcomes challenges associated with complex nucleic acid structures and probe hybridization.