The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Mike G Makrigiorgos - One of the best experts on this subject based on the ideXlab platform.

  • cold pcr enhanced high resolution melting enables rapid and selective identification of low level unknown mutations
    Clinical Chemistry, 2009
    Co-Authors: Coren A Milbury, Mike G Makrigiorgos
    Abstract:

    Background: Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature–PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples. Methods: Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6–8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced. Results: Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons. Conclusions: COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Jin Li, Lilin Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Lili Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

W J Davies - One of the best experts on this subject based on the ideXlab platform.

  • rapid low temperature induced stomatal closure occurs in cold tolerant commelina communis leaves but not in cold sensitive tobacco leaves via a mechanism that involves apoplastic calcium but not abscisic acid
    Plant Physiology, 2001
    Co-Authors: Sally Wilkinson, Alison Lee Clephan, W J Davies
    Abstract:

    Commelina communis stomata closed within 1 h of transferring intact plants from 27°C to 7°C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl2 by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm3) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.

Pasi A Janne - One of the best experts on this subject based on the ideXlab platform.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Jin Li, Lilin Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Lili Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Lili Wang - One of the best experts on this subject based on the ideXlab platform.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Lili Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Jin Li - One of the best experts on this subject based on the ideXlab platform.

  • coamplification at lower denaturation temperature pcr increases mutation detection selectivity of taqman based real time pcr
    Clinical Chemistry, 2009
    Co-Authors: Jin Li, Lilin Wang, Pasi A Janne, Mike G Makrigiorgos
    Abstract:

    Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.