DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms

Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this 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 melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations 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 homoduplex 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. © 2001 American Association for Clinical Chemistry Detection of variations in the canonical sequence of the human genome is a cornerstone for the analysis of genetic diseases, genetically influenced traits, and the origin and structure of populations. Single nucleotide polymorphisms (SNPs) 1 comprise the most abundant category of DNA sequence variation, occurring at a rate of ;1 per 500 nucleotides in coding sequences and at a higher rate in noncoding sequences (1 ). Genome wide there are several million common SNPs. SNPs are highly amenable for high-throughput genotyping using efficient methodologies, including DNA arrays (2‐ 4) , mass spectrometry (5 ), and PCR end-plate read methods such as TaqMan (PE 7700 Sequence Detector) (6 ). For this reason, detection of SNPs of moderate or high abundance (i.e., with rare allele frequencies of .10%) has been made a high priority and may be successfully accomplished by analyzing a relatively small complement of chromosomes. Sequencing of 10 chromosomes (five persons) detects approximately two-thirds of all sequence variants with a frequency of 10%. However, SNPs that alter gene expression or affect structure of the gene product often are rare. Sequencing of 10 chromosomes detects ,10% of sequence variants with a frequency of 1%, a frequently used definition for polymorphism. The continuing requirement for detection of rare SNPs will maintain the need for high-throughput, inexpensive methods for sequence variant detection. Sequence variant detection methods access physical properties of DNA. Several of the most powerful methods for SNP detection [e.g., denaturing gradient gel electrophoresis (DGGE) and denaturing HPLC (dHPLC)] are ultimately based on the thermodynamic properties of DNA duplexes or singlestranded DNA [i.e., single strand conformational polymorphism (SSCP) analysis]. For many years it has been understood that the relative thermostability of a DNA duplex increases with increas