David C Willoughby

US Patent:

20070031865, Feb 8, 2007

Filed:

Jul 6, 2006

Appl. No.:

11/456082

Inventors:

David Willoughby - Jupiter FL, US

International Classification:

C40B 30/06
C40B 40/08

US Classification:

435006000

Abstract:

The invention is directed to processes for constructing DNA Libraries in which ssDNA containing a chemical modification (CM) at or near the 5′- or 3′-terminus is prepared from a RNA or DNA source, a 1universal oligonucleotide (Oligo A′) is ligated to the 3′-of the ssDNA, and a 2universal oligonucleotide (Oligo B) is ligated to the 5′-terminus of the ssDNA. Chemical modifications useful for the process are functional groups capable of binding a solid support with high affinity, or functional groups that can mediate a non-enzymatic ligation. In one embodiment of the invention, a CM at or near the 5′-terminus of the ssDNA mediates binding of the ssDNA to a solid support, allowing removal of residual unligated Oligo A′ prior to ligation of Oligo B. In another embodiment of the invention, a CM at or near the 5′-terminus of ssDNA mediates non-enzymatic ligation of Oligo B to the 5′-terminus of ssDNA, under conditions in which no further ligation of Oligo A′ can occur. Libraries prepared by the method of the invention can be directly amplified by PCR or other methods. Amplified libraries, derived from minute quantities of RNA and DNA, can be used in gene expression studies, analysis of DNA polymorphisms, and high throughput sequencing. Methods of attaching the finished DNA Libraries to a solid supports for archiving are also disclosed. The invention further provides kits for carrying out the processes of the invention.