Dietmaier, Wolfgang and Hartmann, Arndt and Wallinger, Sabine and Heinmoeller, Ernst and Kerner, Thomas and Endl, Elmar and Jauch, Karl-Walter and Hofstaedter, Ferdinand and Rueschoff, Josef (1999) Multiple mutation analyses in single tumor cells with improved whole genome amplification. AMERICAN JOURNAL OF PATHOLOGY, 154 (1). pp. 83-95. ISSN 0002-9440,
Full text not available from this repository. (Request a copy)Abstract
Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally,, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12, of Ki-ms by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR, The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA. from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 3%) and five cells (efficiency rate I-PEP-PGR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 50 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses In diagnostic and experimental tumor pathology.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | POLYMERASE CHAIN-REACTION; P53 GENE-MUTATIONS; ULTRAVIOLET-RADIATION FRACTIONATION; RHD BLOOD-TYPE; MICROSATELLITE INSTABILITY; PREIMPLANTATION DIAGNOSIS; CYTOGENETIC ANALYSIS; PCR AMPLIFICATION; CANCER-PATIENTS; BLADDER-CANCER; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Chirurgie Medicine > Lehrstuhl für Pathologie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 23 Nov 2022 06:40 |
| Last Modified: | 23 Nov 2022 06:40 |
| URI: | https://pred.uni-regensburg.de/id/eprint/48595 |
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