Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Kyger, Erich M.; Krevolin, Mark D.; Powell, Michael J.

doi:10.1006/abio.1998.2687

Cited by 66 publications

(42 citation statements)

References 22 publications

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“…In the study of these authors PCR clamping followed by SSCP analysis allowed detection of p53 gene mutations even in samples with a 200-fold excess of wild-type genes. Similar to our data, most PCR clamping protocols result in specific binding of PNA oligomers at 68 or 70°C (2,3,12,16,21). The fact that binding of PNA oligomers is less sensitive to base composition and length of the oligomer definitely simplifies the design of PCR clamping experiments.…”

supporting

confidence: 84%

Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement in the Determination of Microbial Diversity

Wintzingerode

Landt

Ehrlich

et al. 2000

Appl Environ Microbiol

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Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.

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supporting

confidence: 84%

Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement in the Determination of Microbial Diversity

Wintzingerode

Landt

Ehrlich

et al. 2000

Appl Environ Microbiol

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“…LightCycler is one of the newly developed equipments that feature rapid thermal cycling and flexible real-time detection. [4][5][6][7][8][9][10] Furthermore, microchips for PCR have also been developed and shown superiority in fast speed, high sensitivity, and being easy to carry. [11][12][13][14][15][16][17][18] After the amplification of specific fragments, the confirmation of PCR products is indispensable.…”

Section: Introductionmentioning

confidence: 99%

Rapid Microvolume PCR of DNA Confirmed by Microchip Electrophoresis

et al. 2005

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PCR is an indispensable technique used in DNA analysis. However, with the traditional methods, the time spent on amplification and the subsequent analysis of PCR products is generally long. Therefore, it is essential to improve these two steps so that the whole procedure can be made faster. In the present work, with λ-DNA as the control template, the amplification of 300-bp fragment could be completed within 37 s with capillary reaction chambers of LightCycler, and the following analysis of PCR products could be completed within 120 s with microchip electrophoresis as the detector. Since the high detection sensitivity of microchip electrophoresis, PCR products with template concentration as low as 5 fg/µL could be detected only after 435 s of amplification. In addition, based on additional optimized conditions simulated by CoventorWare, PCR microchips with distinct structure of the reaction chambers have been designed and successfully applied to the amplification of 300-bp fragment. By comparison, those chambers with ellipse and racket shapes were found to offer very high amplification efficiency. All of these results demonstrate the promise of integrating PCR and electrophoresis on microchip for developing easy-carrying instruments for the fast in situ detection of DNA.

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“…For instance, PNA clamping could be used to improve the selective amplification of closely similar sequences by allele specific oligos (7) or as a prelude to subsequent detection of allelic variants by SSCP (6). Likewise, it was possible to combine the clamping method with real-time fluorescence detection of amplicons by a double strand DNA-selective dye in a closed system (13). Given the trend towards such formats in the diagnostic industry this demonstration is of significant importance.…”

Section: Perspectivesmentioning

confidence: 99%

PCR Clamping

2000

Current Issues in Molecular Biology

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An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases. IntroductionMethods that facilitate the rapid detection of single base pair mutations in DNA are of considerable importance in DNA diagnostics and the emerging field of pharmacogenetics. As the diagnostic industry seeks to avoid problems with amplicon contamination by combining amplification and detection in a closed system, it is furthermore desirable that such mutation detection methods are compatible with these new formats. A popular method is to use allele specific primers in the amplification reaction. As shown by Kwok et al.(1), however, most 3' mismatches between a primer and its template do not significantly impair the PCR reaction.To improve this situation we have developed a method that enhances the specificity of the PCR reaction by targeting the initial step involved in non-specific amplification (2), i.e., the binding of the primer to a mis-PCR Clamping matched target sequence. As shown in Figure 1, the method operates by competition for a common target site between a PNA (complementary to the wild-type target sequence) and one of the PCR primers (complementary to the mutant target sequence), or vice versa. When the template contains the wild-type sequence, PNA binding will dominate over primer binding due to the higher affinity of the matched PNA for the target site. As PNA cannot be extended by the Taq-polymerase the effect of this binding is that the amplification reaction is impaired. When the mutant sequence is present, PCR primer binding will dominate over PNA binding with the resulting generation of amplicons.PCR clamping can also operate by interfering with primer elongation (2). In one set-up the PNA is located at a distance from the PCR primer. In this case, clamping is expected to operate by elongation arrest. In another setup the PNA is located adjacent to one of the PCR primers. Here, clamping is expected to operate by preventing initiation of primer elongation.In order for the clamping reaction to function efficiently the fully complementary PNA must bind to its target sequence ahead of binding of the mismatched DNA primer, or vice versa. The most important variables that affects this ordered addition of oligos are the Tm of the PNA and DNA for their respective target sites, the concentration of the PNA and DNA primer and the kinetics of PNA and DNA hybridization. In order to reduce the effects of PNA concentration and kinetics we expanded the normal 3 step PCR cycle with a PNA annealing step which is set at a temperature where only the fully complementary PNA can bind to its target site (Figure 2). 28 Ørum An important aspect of the clamping method is that it does not require complete blocking of all the target sites in each cycle to work efficiently. In the set-up shown in Figure 1, w...

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Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Cited by 66 publications

References 22 publications

Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement in the Determination of Microbial Diversity

Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement in the Determination of Microbial Diversity

Rapid Microvolume PCR of DNA Confirmed by Microchip Electrophoresis

PCR Clamping

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