Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification. |
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| Authors: | H Z Peng P G Isaacson T C Diss L X Pan |
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| Affiliation: | Department of Histopathology, University College, London Medical School. |
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| Abstract: | AIM--To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples. METHODS--Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. RESULTS--Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA. CONCLUSION--This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available. |
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