METHOD FOR AMPLIFYING NUCLEIC ACIDS

• US 20130210008A1
• Filed: 08/31/2011
• Published: 08/15/2013
• Est. Priority Date: 09/01/2010
• Status: Active Grant

• Current Assignee: Koninklijke Philips Electronics N.V. (Koninklijke Philips N.V.)
• Original Assignee: Koninklijke Philips Electronics N.V. (Koninklijke Philips N.V.)
• Inventors: Feitsma, Harma, Van Der Zaag, Pieter Jan, Overdijk, Marlieke, Van De Stople, Anja
• Granted Patent: US 9,005,896 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present invention describes methods for amplifying a target nucleic acid wherein target nucleic acids hybridise to capture probe nucleic acids which are immobilized to a support via their 5′ end. The hybridization product is further extended with a polymerase to form a double stranded nucleic acid. To this double stranded nucleic acid, a hairpin nucleic acid is ligated. This ligation product is further amplified and sequenced.

Claims

1. A method for amplifying a target nucleic acid comprising the steps of:

a) providing a support with a plurality of a nucleic acid capture probe, wherein said plurality of probes is immobilized to the support via the 5′

end of the nucleic acid,

b) hybridizing a target nucleic acid to the capture nucleic acid probe to form a probe/target complex,

c) extending the capture probe nucleic acid with a polymerase wherein the target nucleic acid is used as template for the extension of the capture probe nucleic acid,

d) ligating the extended target/probe double stranded nucleic acid to a hairpin nucleic acid wherein the 5′

end of the hairpin nucleic acid is ligated to 3′

end of the extended probe and wherein the 3′ and

of the hairpin nucleic acid is ligated to the 5′

end of the target nucleic acid,

e) allowing the 3′

end of the target nucleic acid to bind to a further probe of said plurality of nucleic acid capture probes on said support,

f) extending the 3′

end of said further probe by a polymerase,

g) amplifying the nucleic acid obtained in step f) by repeating steps e) and f).

2. The method according to claim 1, wherein the polymerase in step c) has a 3′

to 5′

proofreading exonuclease activity and wherein the hairpin nucleic acid forms a blunt ended stem.

3. The method according to claim 1 or 2, wherein the polymerase in step c) is Pfu (Pyrococcus furiosus) DNA polymerase.

4. The method according to claim 1, wherein the polymerase in step c) has a 3′

terminal transferase activity, resulting in an overhang at the 3′

end of the double stranded nucleic acid and wherein the hairpin nucleic acid has a 5′

overhang, complementary to the 3′

of the double stranded nucleic acid.

5. The method according to claim 1 or 3 wherein the polymerase in step c) is Taq (Thermophilus aquaticus) DNA polymerase, generating a 3′

overhang of adenine and wherein the 5′

overhang of the hairpin is thymidine.

6. The method according to claim 1, wherein the polymerase in step c) is Klenow polymerase, which is used in the presence of a single type of nucleotide generating a series of identical nucleotides along the captured fragment sequence.

7. The method according to claim 6, wherein said single type of nucleotide is dTTP.

8. The method according to claim 1, wherein said ligating the extended target/probe double stranded nucleic acid to a hairpin nucleic acid of step d) is performed in the presence of PEG8000 in the ligation reaction.

9. The method according to any one of claims 1 to 8, wherein the target nucleic acid is DNA.

10. The method according to any one of claims 1 to 9, wherein the hairpin sequence comprises in its loop a sequence for a rare cutting restriction enzyme.

11. The method according to any one of claim 1 to 10, where the support is a planar support, comprising different distinct zones, each zone comprising a plurality of a different probe.

12. The method according to any one of claims 1 to 10, wherein the support is a microcarrier, wherein said microcarrier comprises a plurality of one nucleic acid capture probe.

13. The method according to claim 12, wherein the microcarrier comprises a detectable label and wherein the detectable label defines the sequence of the capture probe attached to the microcarrier.

14. The method according to any of claims 1 to 13, wherein the amplification in step g) of claim 1 is followed by a sequence determination of the nucleic acid corresponding to the target nucleic acid.

15. The method according to claim 14, wherein the sequence determination is performed on a planar support or a labeled microcarrier of claim 13, further comprising the step of correlating the determined sequence with the zone on the planar support or the label on the microcarrier.