Random strand transfer recombination (RSTR) for homology-independent nucleic acid recombination

Random strand transfer recombination (RSTR) relies on the ability of viral reverse transcriptases to undergo homology-independent template switches during DNA synthesis. To facilitate strand transfer events single stranded template DNA was hybridized via noncoding complementary sequence stretches which flank the genes. The resulting bulb shaped heteroduplex was used as template for the reverse transcriptase driven DNA polymerization in which template switch events occur spontaneously. Switches were promoted by induced pausing of the viral polymerase at digoxygenin labeled nucleotides. RSTR of homologous genes was demonstrated by the recombination of two genes encoding (R)-hydroxynitrilase isoenzymes from Prunus amygdolus. For non-homologous RSTR we recombined the genes of the Havea brasiliensis (S)-hydroxynitrilase and the estC gene from Burkholderia gladioli. Base-pairing dependent recombination took place spontaneously and at high frequency between genes with low and high sequence homology.