Primer-extension pre-amplification of DNA from paraffin-embedded tissue: Analysis of concurrent breast lesions
- Univ. of Texas Health Science Center, San Antonio, TX (United States); and others
We are examining concurrent breast lesions (proliferative disease, in situ cancers and invasive cancers) that co-exist in the same breast for clonality studies of breast cancer evolution. The archival tissue samples with concurrent breast lesions suitable for these analyses are formalin-fixed, paraffin-embedded tissue blocks. We have successfully implemented microdissection and DNA extraction protocols from lesion/normal tissue sets from breast cancer patients. These preparations permit the efficient polymerase chain reaction amplification of highly polymorphic simple sequence repeat polymorphisms (SSRPs) for the genetic analysis. However, the unequal yield of DNA from particularly small lesions and/or the paucity of normal breast ductal epithelium has made extensive testing of these samples difficult. In order to equalize the recovery of DNA from the various morphologically defined lesions and normal tissue, we have implemented the method of primer-extension pre-amplification (PEP). In order to demonstrate the fidelity of the PEP technique in this application, we selected a series of 16 paired normal-lesion DNAs from lysates of formalin-fixed, paraffin-embedded tissues. These samples had been previously analyzed for loss-of-heterozygosity by direct PCR amplification with SSRPs. The PEP products were re-amplified with the locus specific SSRPs to determine whether the genetic characteristics of the tumor and normal samples were preserved. For the eight SSRP loci tested thus far, all of the samples amplified and showed the same patterns of LOH as seen before. These results suggest that PEP may be a generally useful technique for genetic analysis of microscopic neoplastic lesions. At least 20-fold amplification of template DNA was observed in our experiments. We expect that this technique will also permit analysis of DNA from formalin-fixed, paraffin-embedded tissue by comparative genomic hybridization.
- OSTI ID:
- 133555
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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