Method for amplifying specific nucleic acids in parallel

• US 7,883,849 B1
• Filed: 03/31/2005
• Issued: 02/08/2011
• Est. Priority Date: 05/18/2004
• Status: Active Grant

• Current Assignee: Olink AB
• Original Assignee: Olink AB
• Inventors: Dahl, Fredrik
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US11/596,972
• Time in Patent Office: 2,140 Days
• Field of Search: None
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites

Abstract

A method for amplifying a plurality of target sequences that minimizes amplification artefacts is provided. A sample of interest is fragmented into fragments, where each fragment that includes a target sequence has at least one defined end sequence. Selector constructs, all comprising a primer pair motif and each individual selector comprising one or two protruding ends complementary to the defined end sequences of the fragments containing the target sequences, are brought in contact with the fragments. After ligation, the selected target sequences are amplified in parallel using a primer-pair specific for the primer-pair motif common to the selectors.

Claims

1. A method of amplifying a plurality of target sequences in a nucleic acid sample, comprising the steps:

(a) fragmenting the nucleic acid sample into fragments wherein each fragment that includes a target sequence has at least one defined end sequence,

(b) denaturing the nucleic acid sample, if the nucleic acid sample is double-stranded, to produce single-stranded fragments

(c) bringing the single-stranded fragments in contact with a plurality of (i) one or (ii) two double-stranded selector constructs, wherein;

in (i) said selector construct comprises a long oligonucleotide and a short oligonucleotide hybridized thereto such that the long oligonucleotide contains two protruding end sequences complementary to the defined end sequences, or complementary to one defined end sequence and one defined internal sequence, of the target-containing nucleic acid fragments, and wherein both oligonucleotides contain at least one primer motif such that the selector construct contains a primer-pair motif; and

in (ii) said two selector constructs each comprise a long oligonucleotide and a short oligonucleotide hybridized thereto such that the long oligonucleotides each contain one protruding end sequence, said protruding end sequences being complementary to each defined end sequence of the target-containing nucleic acid fragment, and wherein the long oligonucleotides and the short oligonucleotides contain at least one primer motif such that the selector constructs contain a primer-pair motif,

(d) allowing the protruding end sequences of the selectors to hybridized with the fragments,

(e) joining the selectors and fragments by ligation, and

(f) amplifying the selected target sequences in parallel using a primer-pair specific for the primer-pair motif common to at least a subset of the selectors, which subset has a unique primer-pair motif,

wherein, if in (c)(i) a protruding end sequence of the selector construct is complementary to a defined internal sequence of the target-containing nucleic acid fragment, after hybridization (d) the protruding end of the target-containing nucleic acid fragment is cleaved by an endonucleolytic enzyme at the hybridization duplex position before ligation (e).

2. The method according to claim 1, wherein all selectors comprise a common primer-pair motif.

3. The method according to claim 2, wherein the endonucleolytic enzyme is a FLAP endonucleolytic enzyme.

4. The method according to claim 3, wherein the fragmentation of the nucleic acid sample is accomplished using at least one restriction enzyme.

5. The method according to claim 4, wherein the amplification is performed by PCR.

6. The method according to claim 1, wherein the endonucleolytic enzyme is a FLAP endonucleolytic enzyme.

7. The method according to claim 1, wherein the fragmentation of the nucleic acid sample is accomplished using at least one restriction enzyme.

8. The method according to claim 2, wherein the fragmentation of the nucleic acid sample is accomplished using at least one restriction enzyme.

9. The method according to claim 2, wherein the amplification is performed by PCR.

10. The method according to claim 1, wherein the amplification is performed by PCR.

11. The method according to claim 3, wherein the amplification is performed by PCR.

12. The method according to claim 1 (c)(i), wherein remaining linear DNA fragments are removed by enzymatic digestion after ligation of the selectors and fragments to form circular molecules.

13. The method according to claim 1, further comprising an analysis of the generated amplification products using gel electrophoresis, hybridization arrays, sequencing by synthesis, liquid chromatography and/or mass spectrometry.

14. The method according to claim 1, further comprising the step of analyzing genetic variability, identifying microorganisms, analyzing expression, sequencing a genome and/or measuring DNA copy number.