Detection of Genetic Point Mutations by Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping Using Paraffin-Embedded Specimens

Myal, Yvonne; Blanchard, Anne; Watson, Peter H.; Corrin, Michael; Shiu, Robert P. C.

doi:10.1006/abio.2000.4755

Cited by 13 publications

(5 citation statements)

References 9 publications

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“…As far as sensitivity is concerned, most of the various methods can detect somewhere in the range of one mutant allele in the presence of approximately a 1000fold excess of normal alleles. 30,47,51 The highest sensitivity was reported by Sun and colleagues, 52 who were able to detect as few as 3 mutant alleles in the presence of 10,000 normal alleles. Our results are within this range, the method consistently allowing detection of at least 1 mutant allele among 2000 normal alleles, in some cases we were able to identify 1 mutant allele among 20,000 normal alleles.…”

Section: Discussionmentioning

confidence: 98%

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Sotlar

Escribano

Landt

et al. 2003

The American Journal of Pathology

195

180

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The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

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Section: Discussionmentioning

confidence: 98%

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Sotlar

Escribano

Landt

et al. 2003

The American Journal of Pathology

195

180

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“…Shiu et al [3], for the first time demonstrated the mechanism of action of PIP in breast cancer development, which is mediated by over-expression of prolactin. The peptide based nucleotide method (highly sensitive) has clearly established a marked relation between PIP expression and breast cancer development [98]. In earlier days, PIP was considered a biomarker only for breast cancer because in immunohistochemical analysis and in-situ mRNA hybridization experiments, it had been observed that the mRNA of PIP was not detected in a small number of human tumors such as prostate, bladder, colon, uterus, thyroid, and kidney cancer, however, it was detected only in some benign breast tissue [1,99].…”

Section: Tumor Progression and Biomarkermentioning

confidence: 99%

Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications

Hassan

Waheed

Yadav

et al. 2008

Cell. Mol. Life Sci.

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Prolactin inducible protein (PIP) is a 17- kDa single polypeptide chain, known by various names due to its versatile nature and function in human reproductive and immunological systems. It is expressed in several exocrine tissues such as the lacrimal, salivary, and sweat glands. Its expression is up regulated by prolactin and androgens, and estrogens down regulate it. Due to its over-expression in metastatic breast and prostate cancer, presently PIP is considered as a prognostic biomarker. Moreover, its aspartyl-proteinase nature suggests its role in tumor progression. PIP has unique features because it is small in size and plays multiple important functions. Its ability to bind potentially with CD4-T cell receptor, immunoglobulin G (IgG), actin, zinc alpha2-glycoprotein (ZAG), fibronectin and enamel pellicle, reveals its important biological functions. This is the first comprehensive review on the structure and functional analysis of PIP and its clinical applications.

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“…PNA can also bind specific dsDNA targets by triplex formation, even in biological buffers that suppress triplex formation between ssDNA and dsDNA ( , ). Leveraging these unique DNA-binding properties, PNAs have been implemented in platforms for DNA sensing ( − ) and separation ( − ), primarily by linking PNAs to solid surfaces or fluorophores.…”

Section: Introductionmentioning

confidence: 99%

Peptide Nucleic Acid (PNA) Amphiphiles: Synthesis, Self-Assembly, and Duplex Stability

2004

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Peptide amphiphiles comprising a class of conjugates of peptide nucleic acid (PNA), natural amino acids, and n-alkanes were synthesized and studied. These PNA amphiphiles (PNAA) self-assemble at concentrations between 10 and 50 muM and exhibit water solubilities above 500 muM. The highly specific, stable DNA binding properties of PNAs are preserved by these modifications, with no significant differences between the thermodynamics of DNA binding of the PNA peptide and the PNA amphiphile. Proper solubilization of the PNAA required the attachment of (Lys)(2) and (Glu)(4) peptides to PNAs, which affected the PNAA-DNA duplex stability by electrostatic interactions between these charged amino acids and the negatively charged DNA backbone. These electrostatic effects did not affect the specificity of DNA binding, however. Electrostatic effects are screened with added salt, in a manner consistent with previous studies of PNA-DNA duplex stability and predictions from a charged-cylinder model for the duplex.

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Detection of Genetic Point Mutations by Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping Using Paraffin-Embedded Specimens

Cited by 13 publications

References 9 publications

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications

Peptide Nucleic Acid (PNA) Amphiphiles: Synthesis, Self-Assembly, and Duplex Stability

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