DNA amplification and sequencing using DNA molecules generated by random fragmentation

摘要

The present invention is directed to methods to prepare a DNA molecule or a plurality of DNA molecules by random fragmentation. In some embodiments, the present invention regards preparing a template for DNA sequencing by random fragmentation. In specific embodiments, the random fragmentation comprises chemical fragmentation, mechanical fragmentation, or enzymatic fragmentation. In further specific embodiments, a universal sequence is attached to the 3′ end of the DNA fragments, such as by ligation of an adaptor sequence or by homopolymeric tailing with terminal deoxynucleotidyltransferase. In other embodiments, a library is prepared with methods of the present invention.

主权项

1. An in vitro method of incorporating a 3′ OH group onto the 3′ end of a nucleic acid molecule having a 3′ end that lacks an OH group, the method comprising the steps of:
a) obtaining a nucleic acid molecule having a 3′ end that lacks a 3′ OH group; and b) incorporating a 3′ OH group onto the 3′ end that lacks an OH group by exposing the molecule to a terminal deoxynucleotidyltransferase in the presence of a guanine ribonucleotide, guanine deoxyribonucleotide, or both, thereby generating a conditioned nucleic acid molecule.