Selectively primed adaptive driver RDA (SPAD-RDA): an improved method for subtractive hybridization.

Abstract

A modification of the Representational Difference Analysis (RDA) method for subtractive hybridization, termed Selectively Primed Adaptive Driver (SPAD) RDA, is described. It differs from conventional RDA primarily in the manner by which initial driver (D) and tester (T) amplicon complexities are determined, and by optimizing the composition of D with respect to T for each round of subtraction. Total nucleic acid is extracted from serum or plasma and converted to double-stranded DNA/cDNA. A polymerase chain reaction (PCR) primer containing a selective nucleotide(s) at its 3'-end is used to generate amplicons of reduced complexity. Parallel subtractions are carried out, D vs. T (DT) for enrichment of tester-unique sequences and D vs. D (Driver Control or DC) to generate an optimized driver for use in the subsequent round. Following each round, agarose gel electrophoresis is used to visually identify any DT-unique bands through a side-by-side comparison of DT and DC subtraction products. In comparison to conventional RDA, SPAD-RDA achieved greater enrichment of viral sequences from an HCV infected chimpanzee, resulting in isolation of 13.7% of the viral genome, and an overall enrichment for HCV sequences of 239-fold. Virus fragments were also obtained from an HCV-infected human sample subtracted against non-paired human driver sequences. J. Med. Virol. 71:150-159, 2003.