MULTIPLE PCR ANALYSES ON TRACE AMOUNTS OF DNA EXTRACTED FROM FRESH AND PARAFFIN WAX EMBEDDED TISSUES AFTER RANDOM HEXAMER PRIMER PCR AMPLIFICATION

Aim-To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limite d histopathological samples. Methods-Trace amounts of genomic DNA extr acted from fresh tissue and individual lymphoid follicles microdissect ed from archival paraffin wax tissue sections were amplified using a t wo-phase PCR protocol with random hexamers as primers (RP-PCR). The ra ndomly amplified DNA samples were used as templates for specific PCR a mplifications. To check the fidelity of the RP-PCR, products of the sp ecific PCR amplifications were further analysed by single stranded con formation polymorphism (SSCP) or sequencing. R Results-Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including tho se for P globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7- 9), and rearranged immunoglobulin heavy chain gene fragments (VH frame work 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specif ic 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 t issue is available.