Patent Evaluation: Nucleic acid amplification of a target sequence by strand displacement amplification

Abstract

Novelty: A variation of a nucleic acid amplification of a target sequence is described. The method is capable of detecting small amounts of a target sequence in the presence of a large excess of non-target DNA. It could be used for the detection of microorganisms, viruses or mutant oncogenes, such as Mycobacterium tuberculosis, in the presence of human DNA.

Biology: The method relies on the strand displacement activity of a DNA polymerase, such as exo minus Klenow fragment of DNA polymerase I. Four primers are added, two for each strand and two of which contain a HincII site at the end. During DNA synthesis in the presence of dATP aS (and the other three normal nucleotides) and HincII, a HincII site is formed by the incorporation of the appropriate sequences. HincII is added to the reaction and can only nick the strand containing the HincII site. This causes a nick which can be displaced by the Klenow fragment and so many such cycles of incorporation, nicking and strand displacement can continue. Other restriction enzymes such as NciI can be used for this technique.