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- Howell, WM, et al.
(författare)
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iFRET: an improved fluorescence system for DNA-melting analysis
- 2002
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Ingår i: Genome research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 12:9, s. 1401-1407
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.
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| 2. |
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| 3. |
- Prince, JA, et al.
(författare)
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Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation
- 2001
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Ingår i: Genome research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 11:1, s. 152-162
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Tidskriftsartikel (refereegranskat)abstract
- We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by ∼30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.
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