Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To correlate molecular changes with clinical information in prostate tissue, it is necessary to have accurate methods for screening for mutations in clinically available specimens. We have refined the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere with SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samples are heterogeneous in containing tumor, normal tissue, and hyperplastic tissue. To address the first issue, the method included an initial selection of PCR products using exonuclease I, followed by a second-step selection using nested PCR. This step ensures adequate amplification of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue, in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restriction enzyme digestion of products prior to SSCP analysis. Detection of mutations in heterogeneous tissue was evaluated by determining the minimal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous clinical samples, we performed study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and two metastases) and detected abnormally migrating products in six of the prostate cancer specimens (four primaries and two metastases). In five of these samples, there was sufficient DNA to perform sequencing, which disclosed single-base change mutations in all five samples.
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PMID:Identification of p53 mutations in archival prostate tumors. Sensitivity of an optimized single-strand conformational polymorphism (SSCP) assay. 895 19

We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.
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PMID:Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices. 2066 55