Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification

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 twophase 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 RPPCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. Results-Using a minute fraction of RPPCR template pool, multiple PCR analyses, including those for β 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.