title: The use of crude tissue section lysates for PCR assessment of gene copy number with human tumour biopsy samples.
abstract: This paper reports the development of experimental procedures for the use of crude tissue section lysates, in conjunction with a non-radioactive polymerase chain reaction-high performance ion-exchange chromatographic (PCR/HPIEX) method, for the quantitative assessment of gene copy number in human biopsy samples. In particular, these methods have been established for the assessment of gene amplification with the FGF-2, FGF-3, FGF-4 and c-erb-B2 genes with the a single copy IFN-gamma gene used as an internal control. In principle, the same procedures could, in general, be simultaneously applied for monitoring the amplification or deletion of other oncogenes as well as normal genes in mammalian cells. Procedures to optimise the precision of the analysis have been examined, including the influence of the oligonucleotide primer concentrations, cycle number, extension temperature, and method of pre-treatment of the tissue sample with proteinase K. The results confirm that crude tissue lysates, rather than purified DNA samples, can be reliably employed, thus extending the scope of non-radioactive PCR/HPIEX methods for the assessment of aberrant gene copy number to biological samples such as tumour biopsy tissues.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: J F
authors.initials: JF
authors.lastname: Schmitt
authors.forename: M T
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D001706:Biopsy; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000235:genetics; D016889:Endometrial Neoplasms / Q000235:genetics / Q000473:pathology; D005069:Evaluation Studies as Topic; D005260:Female; D018628:Gene Dosage; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004273:DNA, Neoplasm; D009841:Oligonucleotides