A Simple Two-Stage PCR Method for Quality Amplification of Degenerate DNA

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissue banks are invaluable to cancer genetics and biomarker discovery. However, FFPE tissue-derived DNA is degenerate. As such, any PCR technique which will give adequate amplification of such DNA to enable downstream applications is important to the characterization of genetic biomarkers and to personalized medicine. The aim of this article is to describe a two-stage PCR method which efficiently amplifies degenerate FFPE tissue-derived DNA. Ampliconenriched templates were produced by a first-stage multiplex PCR which used five or more primer pairs with similar annealing temperature. Subsequently, the enriched products were used as templates for a singleplex PCR which utilized the same PCR cycling parameters and primer pairs as in the multiplex PCR. We compared the amplification of FFPE-derived and cell line DNA by this new protocol and the conventional PCR using the percentage of samples that achieved amplification at a cycle threshold (ct) of 30 or less, the mean of ct values obtained at the end of PCR, as well as our devised amplification efficiency test. Furthermore we compared the amplification of FFPE DNA using this present protocol and the Quick Multiplex Consensus-PCR (QMC-PCR) protocol on the fast programme of the ABI 7500 Fast Real Time PCR instrument. The present two-stage protocol has better sensitivity and amplification efficiency than the conventional PCR in amplifying degenerate DNA templates; and it also amplifies DNA better than the QMC-PCR protocol in fast cycling Real Time PCR programmes.

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

“…(7,8) Recently, studies have found new isolation methods of exosomes from liquid biopsy samples (urine, serum, breast milk, and plasma of blood), (9,10) and several products have been developed to reduce time and cost, increase reproducibility between laboratories, and efficiently concentrate exosomes. (11)(12)(13)(14) However, because mouse models are associated with complications in collecting liquid biopsy samples, such as small sample volumes due to mouse size, there has not been much research on exosomes in this field. In particular, gene expression analysis using mRNA in exosomes has been difficult.…”