Nested PCR-SSCP assay for the detection of p53 mutations in paraffin wax embedded bone tumours: improvement of sensitivity and fidelity

Wang, L.T.; Smith, Anne; Iacopetta, Barry; Wood, David; Papadimitriou, J; Zheng, Minghao

doi:10.1136/mp.49.3.m176

Cited by 8 publications

(10 citation statements)

References 9 publications

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“…Therefore, the degradation of the DNA extracted from these samples and the alterations caused by formalin-DNA interaction lead to partial or total failure of the polymerase chain reaction (PCR) amplification process, resulting in partial or absent STR profiles. Moreover, preextraction procedures to remove paraffin from fixed tissues seem to be an important step in order to optimise the efficiency of the PCR reaction [13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues

et al. 2010

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In certain circumstances, tumour tissue specimens are the only DNA resource available for forensic DNA analysis. However, cancer tissues can show microsatellite instability and loss of heterozygosity which, if concerning the short tandem repeats (STRs) used in the forensic field, can cause misinterpretation of the results. Moreover, though formalin-fixed paraffin-embedded tissues (FFPET) represent a large resource for these analyses, the quality of the DNA obtained from this kind of specimen can be an important limit. In this study, we evaluated the use of tumoural tissue as biological material for the determination of genetic profiles in the forensic field, highlighting which STR polymorphisms are more susceptible to tumour genetic alterations and which of the analysed tumours show a higher genetic variability. The analyses were conducted on samples of the same tissues conserved in different storage conditions, to compare genetic profiles obtained by frozen tissues and formalinfixed paraffin-embedded tissues. The importance of this study is due to the large number of specimens analysed (122), the large number of polymorphisms analysed for each specimen (39), and the possibility to compare, many years after storage, the same tissue frozen and formalin-fixed paraffin-embedded. In the comparison between the genetic profiles of frozen tumour tissues and FFPET, the same genetic alterations have been reported in both kinds of specimens. However, FFPET showed new alterations. We conclude that the use of FFPET requires greater attention than frozen tissues in the results interpretation and great care in both preextraction and extraction processes.

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Section: Introductionmentioning

confidence: 99%

Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues

et al. 2010

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“…Different authors state that a suitable DNA extraction method from FFPET is essential and needs to be chosen in relation to specific endpoints of a precise investigation; for instance, the increased length of amplicon or the increase of effective amplifiable copy number (Wang et al, 1996;Poljak et al, 2000;Gilbert et al,2007b). Therefore diverse nucleic acid extraction methods already described from FFPET, enable the use of DNA extracted for specific approaches: nested PCR-SSCP assay (Wang et al, 1996), RAPD-PCR (Jacobs et al, 2007), real-time quantitative PCR (Gjerdrum et al,2004), Southern blot hybridisation (e.g. Dubeau et al, 1986;Jackson et al, 1990;Rogers et al, 1990), flow cytometry (Leers et al,1999), microarray comparative genomic hybridization (Vékony et al, 2007) and SNP BeadArrays (Oosting et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…which fixative was chosen, fixative pH, and temperature and time of fixation) as well as post-fixation features (i.e. temperature and period of storage) are considered the most important nucleic acid preextraction parameters (Greer et al, 1991;Miething et al, 2006;Wang et al, 1996;Poljak et al, 2000). In order to optimize the quality and quantity of the nucleic acid extraction, pre-extraction procedures to remove paraffin from fixed tissues (e.g.…”

Section: Introductionmentioning

confidence: 99%

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

Santos

Sá

Bastos

et al. 2009

Research in Veterinary Science

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Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present an advanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to prefixation and fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182 bp.

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“…This was based on a method previously described by Wang et al in 1996. 13 It involved two sets of PCR primers for each of the exons amplified. PCR was performed with 0.8 µM forward and reverse of the first primer sets, 200 µM dNTPs, 3 mM MgCl 2 , and 1.25 U of Taq polymerase (Boheringer Mannheim, Mannheim, Germany) in a final reaction volume of 50 µl.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…13 Briefly, 5-10 µl of the PCR product was mixed with 15 µl of 95% formamide stop buVer and denatured at 95°C for 10 minutes. Following denaturation, the sample tubes were plunged into an ice bath for two minutes.…”

Section: Polymorphismmentioning

confidence: 99%

Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma

Mayall

Jacobson²,

Wilkins³

et al. 1998

Journal of Clinical Pathology

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Aims-To attempt to detect p53 gene mutations in the plasma of patients with large bowel carcinoma. Methods-Plasma was collected from 20 control patients with no history of cancer and from 17 patients with large bowel carcinoma. Corresponding tumour and benign lymph node (control) samples for each of the carcinoma patients were obtained from paraYn blocks. A Dukes' stage was determined for each tumour. DNA was extracted from the plasma samples and the paraYn embedded tissue using previously described methods. A nested primer polymerase chain reaction protocol was used for the amplification of exons 5 to 8 of the p53 gene. "Cold" single strand conformational polymorphism (SSCP) was performed on mini gels and silver stained. Abnormal bands were excised, the DNA eluted, and reamplified for automated dye termination sequencing. Any sample showing an apparent mutation was rechecked from the original extracted DNA sample at least three times. Results-p53 gene mutations were not found in the control specimens. They were found in both the primary tumour and the plasma in three cases, in the primary tumour alone in one case, and in the plasma alone in two cases. One of the latter two cases also had metastatic transitional cell carcinoma of the bladder and the other had widespread metastatic deposits. One of the cases with mutant DNA in both the plasma and the primary was a Dukes' stage B tumour. The others were Dukes' C and Dukes' D. Conclusions-p53 gene mutations can be detected in the plasma of some patients with large bowel carcinoma and these are concordant with those in the primary carcinomas. (J Clin Pathol 1998;51:611-613)

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Nested PCR-SSCP assay for the detection of p53 mutations in paraffin wax embedded bone tumours: improvement of sensitivity and fidelity

Cited by 8 publications

References 9 publications

Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues

Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma

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