Method for generating a double stranded nucleic acid with a single stranded overhang

摘要

The present invention provides a method that combines the construction of double-stranded target amplification products with one or two single-stranded overhangs with improved production of such target amplification products. The single-stranded overhang(s) can be used for post-amplification capture and subsequent detection/manipulation. The single-stranded overhang(s) enable capture/detection/manipulation without interference from the complementary strand in the double-stranded target amplification product.

主权项

1. A method comprising the steps of:
a. Providing a template nucleic acid b. Providing a bifunctional oligonucleotide (bfo) comprising a primer/template (pt) region (complementary to part of the template nucleic acid) in its 3′end and a capture region (cr), wherein the pt region and the cr region is separated by a polymerase block (B) c. Mixing the components of steps a-b and providing conditions that allow the pt region of the bfo to anneal to the template nucleic acid and the B to improve the production of amplified template nucleic acid d. Under conditions allowing primer extension, extending the pt region of the bfo e. Thereby generating a double stranded nucleic acid with a single stranded overhang (dso) corresponding to the cr region of the bfo
wherein the polymerase block (B) is a TINA monomer that can be described by the formula: wherein R is selected from the group consisting of arylethynyl, pyreneethynyl, and 1H-phenanthro[9,10-d]imidazol-2-yl.