Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence.
We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are approximately 1fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.
Twist
Charles R
CR
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth SY23 3DD, Wales, UK.
Winson
Michael K
MK
Rowland
Jem J
JJ
Kell
Douglas B
DB
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D019398:Genes, BRCA1; D008904:Miniaturization; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005456:Fluorescent Dyes; D009711:Nucleotides; D020319:Oligodeoxyribonucleotides, Antisense; D012235:Rhodamines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator