The cycling was immediately followed by incubation at 95[degrees]C for 2 min to denature the DNA, followed by 65[degrees]C for 30 min to facilitate HJ formation (branch migration).
We genotyped 80 genomic DNA samples with the newly developed HAS genotyping one-step thermocycling protocol, which allows for PCR and branch migration in a single tube.
We recently described branch migration inhibition (BMI), a homogeneous mutation detection method based on spontaneous BMI in PCR-amplified DNA (1).
The mixture was denatured and subjected to branch migration (2 min at 95 [degrees]C, followed by 30 min at 65 [degrees]C).
When the two arms of this structure are identical (no mutation or SNP is present), strand exchange via spontaneous branch migration leads to its complete dissociation into two duplex molecules, and no signal is observed.
3, filled symbols), all amplified samples were mixed with an equal amount of a sample homozygous for the predominant allele, the mixtures were denatured, and branch migration was repeated.
It detects only heterozygotes, and if finding that more than one allele of a sequence exists is not deemed sufficient, an additional step is required, which consists of adding a reference amplicon that corresponds to one of the two possible homozygotes to each amplified sample and repeating denaturation and branch migration. This step allows identification of homozygotes for the second allele.
Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.
Formation of a single base mismatch impedes spontaneous DNA branch migration. J Mol Biol 1993; 230:413-24.
DNA branch migration. In: Eckstein F, Lilley DMJ, eds.
In addition to his research in homogeneous methods, Ullman has developed both qualitative and quantitative noninstrumented test methods for drugs, microorganisms, and antibodies; a blood typing and antibody screening system based on fluorescent-bead binding to erythocytes; single primer amplification of DNA based on a novel template switching-process during primer extension; PCR-coupled branch migration
inhibition for detection of any mutation within a defined strand of DNA; and many other less-general methods for use in clinical testing.