Sensitive two-stage PCR of p53 genomic DNA exons 5-9.

Kusser, Wolfgang; Levin, D. B.; Glickman, Barry W.

doi:10.1101/gr.2.3.250

Cited by 14 publications

(9 citation statements)

References 8 publications

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“…PCR amplification for the p53 exon4 codon72 variant (Table S1) and the p53 exon5–9 mutation was performed as previously described [15], [16]. Each PCR product was sequenced using the ABI PRISM 3030xl genetic analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

A Compensatory Role of NF-κB to p53 in Response to 5-FU–Based Chemotherapy for Gastric Cancer Cell Lines

et al. 2014

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Despite of remarkable improvement of postoperative 5-FU–based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU–based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.

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Section: Methodsmentioning

confidence: 99%

A Compensatory Role of NF-κB to p53 in Response to 5-FU–Based Chemotherapy for Gastric Cancer Cell Lines

et al. 2014

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“…As the final concentration of DNA extracted was low, nested PCR was used to amplify p53 exons 5 -8 from each sample (Kusser et al, 1993;Yoshida et al, 2003). The whole region between exons 5 and 8 (inclusive) was amplified initially and then subsequently used as the template for amplification of each individual exon.…”

Section: Mutation Analysismentioning

confidence: 99%

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

et al. 2003

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p53 mutations and loss of heterozygosity have been commonly associated with oesophageal adenocarcinoma. In this investigation, the p53 status of a Welsh population of Barrett's-associated oesophageal adenocarcinomas were fully characterised at the gene sequence, chromosomal, mRNA and protein levels. In total, 31 tumours were examined for p53 gene sequence mutations using RFLP with sequencing, allelic loss of the gene was characterised by FISH, mRNA expression by p53 pathway signalling arrays and protein levels by p53 immunohistochemistry. In all, 9.6% of adenocarcinomas harboured p53 mutations, 24% displayed p53 allelic loss and 83% exhibited p53 protein accumulation. Point mutations and deletions of the gene did not coexist within the same samples. All samples containing p53 mutations also displayed positive immunostaining; however; in the majority of cases, p53 protein accumulation developed in the absence of mutations. The gene expression analysis demonstrated no differences in p53 and mdm-2 transcription levels between the p53 immunonegative and immunopositive samples, indicating other mechanisms underlie the proteins' overexpression. In conclusion, p53 mutations and deletions do not appear to be frequent events in oesophageal adenocarcinomas; however, abnormal accumulation of the protein is present in a vast majority of cases. P53 gene mutations are not the primary cause of protein overexpression -an alternative mechanism is responsible for the positive p53 immunohistochemistry detected.

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“…It uses the same primer pairs and cycling parameters for both stages of the protocol [6,7,[16][17][18][19][20].…”

Section: Discussionmentioning

confidence: 99%

A Simple Two-Stage PCR Method for Quality Amplification of Degenerate DNA

O¹

2015

Int J Cancer Clin Res

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Formalin-fixed, paraffin-embedded (FFPE) tissue banks are invaluable to cancer genetics and biomarker discovery. However, FFPE tissue-derived DNA is degenerate. As such, any PCR technique which will give adequate amplification of such DNA to enable downstream applications is important to the characterization of genetic biomarkers and to personalized medicine. The aim of this article is to describe a two-stage PCR method which efficiently amplifies degenerate FFPE tissue-derived DNA. Ampliconenriched templates were produced by a first-stage multiplex PCR which used five or more primer pairs with similar annealing temperature. Subsequently, the enriched products were used as templates for a singleplex PCR which utilized the same PCR cycling parameters and primer pairs as in the multiplex PCR. We compared the amplification of FFPE-derived and cell line DNA by this new protocol and the conventional PCR using the percentage of samples that achieved amplification at a cycle threshold (ct) of 30 or less, the mean of ct values obtained at the end of PCR, as well as our devised amplification efficiency test. Furthermore we compared the amplification of FFPE DNA using this present protocol and the Quick Multiplex Consensus-PCR (QMC-PCR) protocol on the fast programme of the ABI 7500 Fast Real Time PCR instrument. The present two-stage protocol has better sensitivity and amplification efficiency than the conventional PCR in amplifying degenerate DNA templates; and it also amplifies DNA better than the QMC-PCR protocol in fast cycling Real Time PCR programmes.

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Sensitive two-stage PCR of p53 genomic DNA exons 5-9.

Cited by 14 publications

References 8 publications

A Compensatory Role of NF-κB to p53 in Response to 5-FU–Based Chemotherapy for Gastric Cancer Cell Lines

A Compensatory Role of NF-κB to p53 in Response to 5-FU–Based Chemotherapy for Gastric Cancer Cell Lines

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

A Simple Two-Stage PCR Method for Quality Amplification of Degenerate DNA

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