DNA melting analysis for detection of single nucleotide polymorphisms

Background: Several methods for detection of single nucleotide polymorphism s (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heterodup lex DNA. We present a method for detecting SNPs that is directly based on t his principle. Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA m elting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denatura tion, evaluating the effects of fragment length, dye concentration, DNA con centration, and sequence context using four naturally occurring polymorphis ms (three SNPs and a single-base deletion/insertion). Results: DNA melting analysis (DM) was used successfully for variant detect ion, and we also discovered two previously unknown SNPs by this approach. C oncentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6 %. We readily detected differences in the melting temperature between homod uplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution. Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants. (C) 2001 American Assoc iation for Clinical Chemistry.