High multiplex pcr with molecular barcoding
AssigneeQiagen Sciences, LLC
InventorsYexun Wang, Quan Peng
Life of this patent
Estimating the term…
Abstract
The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).
Claims33
A method for amplifying target nucleic acids in a nucleic acid sample, comprising:
- (a) extending each of a plurality of barcode primers (BC primers) to obtain extension products using the target nucleic acids as templates, wherein
- (i) each barcode primer comprises, from 5′ to 3′, a 1st universal primer sequence (US1), a molecular tag sequence (MT), and a 1st target-specific sequence (TS1),
- (ii) a plurality of barcode primers comprise at least 20 different barcode primers, and
- (iii) among the plurality of barcode primers (BC primers), the 1st universal primer sequences (US1) are the same, but the 1st target-specific sequences (TS1) are different;
- (b) separating the plurality of barcode primers that have not been extended in step (a) from the extension products;
- and
- (c) amplifying the extension products of step (b) in the presence of a plurality of limited amplification primers (LA primers) to obtain a plurality of 1st amplification products, wherein
- (i) each limited amplification primer comprises, from 5′ to 3′, a 2nd universal primer sequence (US2) and a 2nd target-specific sequence (TS2), and
- (ii) among the plurality of limited amplification primers, the 2nd universal primer sequences (US2) are the same, but the 2nd target-specific sequences (TS2) are different.
A kit comprising:
- (1) a plurality of barcode primers (BC primers), wherein
- (i) each barcode primer comprises, from 5′ to 3′, a 1st universal primer sequence (US1), a molecular tag sequence (MT), and a 1st target-specific sequence (TS1),
- (ii) a plurality of barcode primers comprise at least 20 different barcode primers, and
- (iii) among the plurality of barcode primers, the 1st universal primer sequence (US1) are the same, the molecular tag sequences (MT) are different, and the 1st target-specific sequence (TS1) are different;
- and
- (2) a plurality of limited amplification primers (LA primers), wherein
- (i) each limited amplification primer comprises, from 5′ to 3′, a 2nd universal primer sequence (US2) and a 2nd target-specific sequence (TS2), and
- (ii) among the plurality of limited amplification primers, the 2nd universal primer sequences (US2) are the same, but the 2nd target-specific sequence (TS2) are different.
-47. (canceled)
Dependencies are inferred from the claim text.