A FRET-based analysis of SNPs without fluorescent probes.
Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.
Takatsu
Kyoko
K
Kankyo Engineering Co., Ltd, 2-1-38 Shiohama, Kisarazu, Chiba 292-0838, Japan.
Yokomaku
Toyokazu
T
Kurata
Shinya
S
Kanagawa
Takahiro
T
D052160:Benzothiazoles; D003959:Diamines; D031541:Fluorescence Resonance Energy Transfer / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013696:Temperature
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I