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.
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA. rlipsky@mail.nih.gov
R H
RH
Lipsky
C M
CM
Mazzanti
J G
JG
Rudolph
K
K
Xu
G
G
Vyas
D
D
Bozak
M Q
MQ
Radel
D
D
Goldman
D020137:Base Pair Mismatch; D052160:Benzothiazoles; D004247:DNA / Q000235:genetics*; D003959:Diamines; D005456:Fluorescent Dyes; D020180:Heteroduplex Analysis / Q000379:methods; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA