Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods

Wartell, Roger M.; Hosseini, Seyed Homayoun; Powell, Sandra; Zhu, Jian

doi:10.1016/s0021-9673(98)00149-6

Cited by 41 publications

(32 citation statements)

References 56 publications

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“…Single-base differences in otherwise homologous DNA fragments can be discriminated provided that the differences are located within the lowest melting domain, 11 and this domain is clearly separated from the onset of duplex to strands dissociation. 24 Both of these conditions can be satisfactorily achieved by the attachment of a higher melting section or a GC-rich sequence to the DNA fragment of interest, thus rendering the detection of virtually all single-base changes possible. 24 Indeed, the melting properties of dsDNA have been widely exploited for the detection of point mutations in such gel-based approaches as denaturation gradient gel electrophoresis and related methods, 11,22,25 as well as in elution-based methods such as high performance liquid chromatography.…”

Section: Discussionmentioning

confidence: 99%

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

Elenitoba-Johnson

Bohling

2001

The American Journal of Pathology

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Salt Lake City, UtahDNA molecules differing by as little as a single-base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of doublestranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA (ϳ20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of

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Section: Discussionmentioning

confidence: 99%

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

Elenitoba-Johnson

Bohling

2001

The American Journal of Pathology

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“…A possible explanation for this phenomenon is the formation of a transition state during the denaturation process, as described in denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis experiments. 29 The single-stranded portion of the partially melted molecules can form branches, which probably have a higher R th value than ssDNA. Another explanation is that the higher R th value is caused by the denaturated, untethered ssDNA strands that interact with the surface, causing a higher insulation of the NCD surface.…”

mentioning

confidence: 99%

Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

Bon¹,

Grinsven²,

Murib³

et al. 2014

IJN

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Conventional neonatal diagnosis of phenylketonuria is based on the presence of abnormal levels of phenylalanine in the blood. However, for carrier detection and prenatal diagnosis, direct detection of disease-correlated mutations is needed. To speed up and simplify mutation screening in genes, new technologies are developed. In this study, a heat-transfer method is evaluated as a mutation-detection technology in entire exons of the phenylalanine hydroxylase (PAH) gene. This method is based on the change in heat-transfer resistance (R th ) upon thermal denaturation of dsDNA (double-stranded DNA) on nanocrystalline diamond. First, ssDNA (single-stranded DNA) fragments that span the size range of the PAH exons were successfully immobilized on nanocrystalline diamond. Next, it was studied whether an R th change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear R th shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, R th was shown to detect prevalent single-nucleotide polymorphisms, c.473G>A (R158Q), c.932T>C (p.L311P), and c.1222C>T (R408W), correlated with phenylketonuria, displaying an effect related to the different melting temperatures of homoduplexes and heteroduplexes.

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“…The TGGE method exploits two physical properties of DNA: the effect of nucleic acid sequence on temperature-induced unwinding of DNA strands and the influence of partial denaturation on electrophoretic mobility. 16 Each point mutation changes the melting temperature of the PCR product and its electrophoretic mobility. Accordingly, DNA molecules with single base pair differences undergo partial denaturation at temperatures clearly lower than the temperature required for strand separation and migrate slower in gel.…”

mentioning

confidence: 99%

“…Accordingly, DNA molecules with single base pair differences undergo partial denaturation at temperatures clearly lower than the temperature required for strand separation and migrate slower in gel. 7,9,13,16 Thus, TGGE enables separation of DNA fragments of the same size but with different melting temperatures resulting from a single base pair difference. The method can also be applied for routine analysis of many samples in a fairly short time.…”

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confidence: 99%

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Application of Temperature-Gradient Gel Electrophoresis for Detection of Prion Protein Gene Polymorphisms in Polish Świniarka Sheep

Jasik

Reichert

2006

J VET Diagn Invest

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Abstract. This study presents preliminary data on the polymorphism in the prion protein gene of Ś winiarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.

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Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods

Cited by 41 publications

References 56 publications

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

Application of Temperature-Gradient Gel Electrophoresis for Detection of Prion Protein Gene Polymorphisms in Polish Świniarka Sheep

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