The driver is left without PCR priming sequences, and therefore cannot be amplified in subsequent steps. The tester amplicon is denatured in the presence of large amounts of driver amplicon and allowed to reanneal slowly. Fragments unique to the tester amplicon will eventually reanneal with each other, while tester fragments that are shared with driver will reanneal with driver fragments that are in excess. Denature tester amplicon in the presence of large amounts of driver amplicon and allow to reanneal. Only sequences unique to tester that reanneal with priming sequences on both ends will amplify exponentially.
Subtractive hybridization is repeated with difference products and driver amplicon.
PCR Amplification of the Difference Products DNA polymerase is added to fill in the ends of each fragment to create double-stranded priming sequences and PCR is then performed in the presence of the new primer, nucleotides and DNA polymerase. Fragments that are present in both tester and driver amplicons will reanneal to form double-stranded molecules that only contain PCR priming sequences on one end and can amplify only linearly. The amplified fragments that remain after subtractive hybridization and PCR amplification are called difference products. The first round of subtractive hybridization and PCR amplification is mainly subtractive with a small enrichment of the target sequences. By repeating two more rounds of subtractive hybridization and PCR amplification, with the difference products and the driver amplicon still in excess, the target sequences can be amplified to levels that can be detected by agarose gel electrophoresis.