Determination of copy number of short tandem repeat using NAD-dependent ligase and pyrosequencing-compatible method

A sensitive and pyrosequencing-compatible method for determining the copy number of the short tandem repeat (STR) is presented in this study. When Escherichia coli ligase catalyzes the ligation of primer and probes complementary to the proper sites of the target DNA template, it converts nicotinamide adenine dinucleotide to adenosine monophosphate (AMP) and nicotinamide. The AMP release level is proportional to the copy number of the STR and can be measured using adenylate kinase, pyruvate kinase, and luciferase. Unlike current standard methods based on electrophoresis, the present assay is sensitive to the point mutation. Furthermore, after determination of the copy number of the tandem repeat using the proposed method, the DNA templates, primer and probes immobilized onto super paramagnetic beads can be washed and pyrosequencing can be applied for the remaining DNA sequencing. This assay is specially efficient to handle a large number of samples because massively parallel tests could be executed in a microplate photometer. Furthermore, it can work with the pyrosequencing for further sequencing like genome sequencing.