Amplification and Analysis of Selected Targets on Solid Supports

摘要

Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. Targets are circularized by hybridization to probes followed by ligation of the ends of the target to form a closed circle. The targets are then used as template for extension of an array bound probe resulting in extended probes having multiple copies of the target. The extended probes can then be analyzed. The methods may be used for genotyping, sequencing and analysis of copy number.

主权项

1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:
(a) fragmenting the nucleic acid sample to obtain target fragments comprising target sequences; (b) mixing the target fragments obtained in (a) with an array of probes arranged in features on a solid support, wherein each feature comprises multiple copies of a target specific array probe having a first probe region that is perfectly complementary to a first target region and a second probe region that is perfectly complementary to a second target region, wherein the first probe region is 5′ of the second probe region and wherein the first target region is 5′ of the second target region, and wherein the probes are attached to the solid support at the 5′ end of the probe, wherein target fragments hybridize to complementary target specific array probes to form probe:target fragment complexes wherein the target fragment hybridizes to the target specific array probe so that the first target region is hybridized to the first probe region and the second target region is hybridized to the second probe region; (c) joining a 5′ end of each target fragment to the 3′ end of the same target fragment to form target circles; (d) extending the array probes using the target circles as a template in a rolling circle amplification reaction, thereby obtaining extended array probes comprising a plurality of copies of the complement of a plurality of the target fragment; and (e) analyzing the copies.