Allelic discrimination by denaturing high-performance liquid chromatography.
Ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles allows the resolution of single-stranded DNA molecules of identical size (<100 nucleotides) that differ in a single base. Allelic discrimination is obtained by injecting short DNA amplicons containing the genetic variants of interest into an adequately preheated mobile phase that results in the instantaneous complete denaturation of the PCR products. All possible transitions and transversions other than C-->G can be typed accurately. The method complements the discovery of single-nucleotide polymorphisms by means of HPLC based heteroduplex detection under partially denaturing conditions and allows their rapid genotyping without the need of adding a reference chromosome.
Oefner
P J
PJ
DNA Sequencing and Technology Center, Stanford University, Palo Alto, CA 94304, USA. oefner@genome.stanford.edu
D000483:Alleles; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers