Directed enrichment of genomic DNA for high-throughput sequencing

Abstract

The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and / or amplify a target polynucleotide are provided.

Description

RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 785,295, filed on Mar. 23, 2006. The entire teachings of the above application are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] Large numbers of amplicons or polymerase chain reaction products (PCR) products are necessary to amplify every exon in a genome. For example, the amplification all of the exons in the human genome would require approximately 250,000 amplicons or PCR products. For each amplicon, a pair of primers needs to be synthesized and an individual PCR reaction needs to be carried out, which is a costly and unwieldy task for the total number of 250,000 amplicons required to sequence all of the exons in the human genome. [0003] Microarray technologies are available that can synthesize very small quantities of up to about 400,000 oligonucleotides on a single solid support. These 400,000 oligonucleotides could potentially comprise 200,000 primer pai...

AI Technical Summary

Benefits of technology

[0005] The present invention provides microarrays of oligonucleotides, e.g., primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or

Problems solved by technology

However, there is no current method to separate the 200,000 primer pairs into 200,000 separate amplification reactions.

Although all 400,000 oligonucleotides can be liberated from the microarray into a single (multiplex) PCR reaction using known methods, the resulting multiplex PCR reaction has little informative value due to the complexity of the amplified products generated and due to numerous artifacts that result from such a multiplex PCR, such as primer-dimer formation (see, e.g., Dahl et al., Nuc. Ac. Res. (2005), 33(8): e71, and references cited therein).

Images