PCR amplification of 40-year-old paraffin-embedded tumor-tissues - comparison of 4 different DNA extraction and purification methods
| Autor: | G Malone, A Haworth, S Kumar, Patricia Kumar, M Schwarz, Wg Wang |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
chemistry.chemical_classification
Cancer Research Chromatography biology Extraction (chemistry) Multiple displacement amplification Proteinase K DNA extraction Molecular biology law.invention chemistry.chemical_compound Enzyme Oncology chemistry law Primer dimer biology.protein DNA Polymerase chain reaction |
| Zdroj: | International journal of oncology. 5(3) |
| ISSN: | 1019-6439 |
| Popis: | Four different methods of DNA extraction from formalin-fixed, paraffin-embedded tissues were compared for their ability to produce DNA suitable as a template for polymerase chain reaction (PCR). Seven paraffin-embedded rhabdomyosarcoma tissue blocks from the 1950s and one from 1960 were treated with the following extraction methods: 1. Proteinase K digestion and phenol/chloroform extraction; 2. Proteinase K digestion followed by boiling to inactivate the enzyme; 3. Proteinase K digestion, addition of Chelex-100, followed by boiling; and 4. Proteinase K digestion and Prep-A-Gene purification. DNA extracted by methods 1 and 2 was degraded, but DNA of high molecular weight was recovered in every sample extracted by methods 3 and 4 - even though some degradation was observed. Extracted DNA was used as a template for PCR amplification of exon 4 of the PAX-3 gene. PCR was successful in 7 out of 8 samples prepared using methods 3 and 4, producing levels of amplified product equivalent to those obtained with control DNA obtained from fresh lymphocytes. Only very weak products were found in samples prepared by methods 1 (2 out of 8) and 2 (4 out of 8). These results indicate that chelation of polyvalent ions (Chelex-100 method) or obviating the need for boiling (Prep-A-Gene) may protect DNA during extraction. |
| Databáze: | OpenAIRE |
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