Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring.

We report here a new mechanism for allelic discrimination--allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added.

Screening for single-nucleotide polymorphisms using branch migration inhibition in PCR-amplified DNA.

Background: New methods are required for the exploration of the human genome by discovering sequence variations. This study evaluated the performance of a new method for screening a large number of samples for several DNA polymorphisms.

Methods: We used a homogeneous method based on inhibition of spontaneous branch migration by any sequence difference between two molecules of PCR-amplified DNA.

Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.

A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures.

Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene.

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene.