Methods for preparing amplifiable DNA molecules

摘要

The present invention concerns isolation, library preparation and selective amplification from a compositionally heterogeneous pool of DNA fragments of a fraction of molecules, such as those originating from promoter CpG islands and characterized by a high GC content. In particular, the process utilizes a heat-induced segregation of DNA molecules into GC-poor, single-stranded molecule fractions and GC-rich, double-stranded molecule fractions, with subsequent enzymatic conversion of the GC-rich, double-stranded DNA molecules into a library, and, optionally, amplification. In specific embodiments, the isolation process is used to generate promoter-enriched genomic and methylome libraries for research and diagnostic applications, for example.

主权项

1. A method of preparing a plurality of amplifiable DNA molecules, comprising:
providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich; subjecting the plurality to a first temperature such that the GC-poor regions are substantially denatured and such that the GC-rich regions are undenatured or are denatured only in part; subjecting the plurality to a second temperature such that at least part of the GC-poor regions incompletely renature and such that at least part of the GC-rich regions substantially completely renature, thereby producing renatured amplifiable GC-rich molecules; ligating an adaptor onto the end of at least some of the renatured GC-rich molecules to produce adaptor-ligated molecules, wherein the adaptor is further defined as a stem-loop oligonucleotide comprising an inverted repeat and a loop.