Locus specific amplification using array probes
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. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.
1. A method for amplifying a plurality of targets, the method comprising:
(a) hybridizing each target to a first complementary support bound probe in an array comprising a plurality of support bound probes, where probes of the same sequence are present in the same feature and wherein different targets hybridize to different first probes in different features;
(b) extending the first probes using the hybridized targets as template to obtain extended first probes;
(c) removing the target;
(d) ligating an adapter to 3′ end of the extended first probes, wherein the adapter comprises a top strand that is attached to the extended first probes, and a bottom strand that is hybridized to the first strand, wherein the adapter comprises an RNA polymerase promoter;
(e) transcribing multiple RNA copies of the extended first probe using RNA polymerase;
(f) allowing the copies of the extended first probe to hybridize to probes of the array in the same feature; and
(g) analyzing the copies to determine the sequence of the targets.
2. The method of claim 1 , wherein the RNA polymerase promoter is a T 7 promoter.
3. The method of claim 1 , wherein transcribing multiple RNA copies of the extended first probe using RNA polymerase includes in vitro transcription.
4. The method of claim 1 , wherein the bottom strand comprises a first chimeric primer.
5. The method of claim 4 , wherein the first chimeric primer is extended to generate a copy of the extended first probe.
6. The method of claim 5 , further comprising digesting the RNA portion using RNase H.
7. The method of claim 4 , further comprising hybridizing a second chimeric primer where the RNA portion was removed and extended to displace a strand.
8. The method of claim 7 , wherein the displaced strand is hybridized to adjacent probes.
9. The method of claim 1 , wherein the top strand of the adaptor is replaced by tailing the extended first probe using at least one of a terminal transferase or a polymerase that adds untemplated nucleotides.
10. The method of claim 9 , wherein the RNA portion is a specific sequence, wherein the specific sequence is a new priming sequence and the tailed extended probe is extended using that sequence as a template for template directed extension such that the complement of the RNA portion is incorporated into the extended probe.
11. The method of claim 1 , further comprising adding a restriction site via the adapter, wherein the restriction site is used to initiate strand-displacement amplification.
12. The method of claim 1 , wherein the adaptor includes a cleavage site.
13. The method of claim 12 , wherein the cleavage site is a restriction site.
14. The method of claim 1 , wherein the adapter includes a primer binding site.