Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Determining the allelic status of multiple single nucleotide polymorphisms (SNPs) is crucial for understanding genetic variation and disease associations.
• Traditional methods can be complex and time-consuming for analyzing multiple SNPs within a haplotype.

Purpose of the Study:

• To develop and validate a rapid, allele-specific method for assigning multiple SNPs to the same haplotype.
• To demonstrate the utility of this method using the glucocorticoid receptor (GR) gene as a model system.

Main Methods:

• Development of an allele-specific long-range PCR/sequencing (ALP/S) method.
• Design of allele-specific primers targeting distal SNPs (intron 4 and exon 9 alpha) of the GR gene.
• Utilizing a two-round PCR approach followed by sequencing to confirm SNP linkage.

Main Results:

• Successfully identified four SNPs, including two novel variations, within the GR gene across investigated cell lines.
• Demonstrated that the four identified SNPs are located on the same allele, forming a haplotype.
• The terminal SNPs were confirmed to be 19 kb apart, showcasing the method's long-range capability.

Conclusions:

• The developed ALP/S method is rapid and effective for determining haplotypes involving more than three SNPs.
• This technique provides a valuable tool for precise genetic analysis and haplotype assignment in various research applications.