Scoring single-nucleotide polymorphisms at the single-molecule level by counting individual DNA cleavage events on surfaces.
Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) of the cleavage products. The ability of RCA to produce single-stranded DNA tens of thousands of nucleotides in length from a single cleaved DNA molecule on the surface suggested the possibility of detecting individual cleavage events on the surface. The feasibility of this approach to SNP scoring is shown here. Individual cleavage events on the surface are detected using fluorescence microscopy to visualize the single-stranded DNA product of the RCA reaction labeled with the fluorescent dye SYBR Green I. The surface density of fluorescent features observed is dependent upon the concentration of target DNA. Future reductions of the sample volume and optimization of the reaction conditions offer the potential of being able to perform such analyses on as little as a single copy of genomic DNA target.
Nie
Bei
B
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Shortreed
Michael R
MR
Smith
Lloyd M
LM
D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D005787:Gene Frequency; D014644:Genetic Variation; D015894:Genome, Human; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009711:Nucleotides / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D013499:Surface Properties
D009711:Nucleotides; D004247:DNA