Single base pair mutation analysis by PNA directed PCR clamping

Ørum, Henrik; Nielsen, Peter E.; Egholm, Michael; Berg, Rolf H.; Buchardt, Ole; Stanley, Christopher B.

doi:10.1093/nar/21.23.5332

Cited by 308 publications

(193 citation statements)

References 18 publications

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“…Heteroplasmy detection requires a very sensitive method able to detect mutations at low levels, hence the selection of PCR with PNA/DNA sequencing method which provides an age threshold for the appearance of the mutation [10,11]. The Peptid Nucleic Acid (PNA) molecule is a DNA mimic, in which the negatively charged sugarphosphate DNA backbone is replaced by an achiral, neutral polyamide backbone formed by repetitive units of N-(2-aminoethyl) glycine.…”

Section: Introductionmentioning

confidence: 99%

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones

Lacan

Thèves

Amory³

et al. 2008

Int J Legal Med

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Abstract:The aim of this study was to demonstrate the presence of the A189G age-related point mutation on DNA extracted from bone. For this, a PNA/DNA sequencing method which can determine an age threshold for the appearance of the mutation was used.Initially, work was done in muscle tissue in order to evaluate the sensitivity of the technique and afterwards in bone samples from the same individuals. This method was also applied to ancient bones from 6 well preserved skeletal remains. The mutation was invariably found in muscle, and at a rate of up to 20% in individuals over 60 years old.In modern bones, the mutation was detected in individuals aged 38 years old or more, at a rate of up to 1%, but its occurrence was not systematic (only 4 out of 10 of the individuals over 50 years old carried the heteroplasmy). For ancient bones, the mutation was also found in the oldest individuals according to osteologic markers. The study of this type of age-related mutation and a more complete understanding of its manifestation has potentially useful applications. Combined with traditional age markers, it could improve identification accuracy in forensic cases or in anthropological studies of ancient populations.

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Section: Introductionmentioning

confidence: 99%

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones

Lacan

Thèves

Amory³

et al. 2008

Int J Legal Med

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“…To detect a minimal amount of mutant DNA in clinical samples, peptide-nucleic-acid (PNA) oligomers have been developed (Orum et al, 1993). PNAs are non-extendable oligonucleotides in which the ribose-phosphate backbone is replaced by 2-aminoethyl glycine units linked by amide bonds.…”

mentioning

confidence: 99%

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

et al. 2009

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Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P <0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR ( P <0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

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“…These PNA/DNA hybrids have greater thermal stabilities than those of DNA/DNA hybrids, and the PNA oligomers cannot be extended by DNA polymerases. 16 LNA is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2= oxygen and the 4= carbon. 17 The ⌬T m s between matched and mismatched PNA/DNA or LNA/DNA duplexes usually exceed 10°C, sufficient for the selection of minor variant alleles with sensitive detection.…”

Section: Discussionmentioning

confidence: 99%

“…17 The ⌬T m s between matched and mismatched PNA/DNA or LNA/DNA duplexes usually exceed 10°C, sufficient for the selection of minor variant alleles with sensitive detection. 16,18 Unlike other commonplace methods, such as amplification refractory mutation system PCR, this method can enrich different base changes on the target sites using a single wildtype-specific PNA/LNA. In principle, this does not aberrantly alter the wild-type sequences and leads to much lower false-positive rates.…”

Section: Discussionmentioning

confidence: 99%

Mutant Enrichment with 3′-Modified Oligonucleotides

Lee

Kim

Kown

et al. 2011

The Journal of Molecular Diagnostics

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Many clinical situations necessitate highly sensitive and reliable molecular assays; however, the achievement of such assays remains a challenge due to the inherent limitations of molecular testing methods. Here, we describe a simple and inexpensive enrichment technique that we call mutant enrichment with 3=-modified oligonucleotides (MEMO). The method is based on the use of a 3=-modified oligonucleotide primer that blocks extension of the normal allele but enables extension of the mutated allele. The performance of the technique was evaluated with respect to its ability to detect common cancer mutations in the EGFR, KRAS, BRAF, TP53, JAK2, and NPM1 genes. We achieved sensitivities of 10 ؊2 to 10 ؊6 using downstream Sanger sequencing, depending on the concentrations and thermodynamics of the primers. MEMO

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Single base pair mutation analysis by PNA directed PCR clamping

Cited by 308 publications

References 18 publications

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

Mutant Enrichment with 3′-Modified Oligonucleotides

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