Primers useful for sizing nucleic acids

• US 6,949,633 B1
• Filed: 08/25/1998
• Issued: 09/27/2005
• Est. Priority Date: 05/22/1995
• Status: Expired due to Fees

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Becker, Christopher Hank, Monforte, Joseph Albert, Pollart, Daniel Joseph, Shaler, Thomas Andrew
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/139,386
• Time in Patent Office: 2,590 Days
• Field of Search: 536/22.1, 536/23.1, 536/24.33, 536/25.32
• US Class Current: 536/22.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The present invention provides modified oligonucleotide primers designed to incorporate a cleavable moiety so that a 3′ portion of the primer (linked to an extension product) can be released from an upstream 5′ portion of the primer. Upon selective cleavage of the cleavable site, primer extension products that contain about five or fewer base pairs of the primer sequence are released, to provide more useful sizing and sequence information per fragment than extension products containing the entire primer.

Claims

1. A nucleic acid primer having a 5′

end and a 3′

end, comprising;

(a) a first region containing the 5′

end of the primer and an immobilization attachment site; and

(b) a second region containing the 3′

end of the primer including a free 3′

hydroxyl and a selectively chemically cleavable site, wherein;

the 3′

end is capable of being extended by an enzyme to generate an extension segment, whereby, when the primer is immobilized via the immobilization attachment site, and the selectively chemically cleavable site is cleaved, the remainder of the primer remains immobilized; and

the selectively chemically cleavable site comprises a modified sugar or a chemically cleavable group incorporated into the phosphate backbone.

2. The primer of claim 1, wherein the selectively chemically cleavable site is located at or within about five nucleotides from the 3′

end of the primer.

3. The primer of claim 2, wherein the second region of the primer comprises a single nucleotide.

4. The primer of claim 3, wherein the second region comprises a ribonucleotide.

5. The primer of claim 1, wherein the selectively chemically cleavable site comprises a chemically cleavable group incorporated into the phosphate backbone.

6. The primer of claim 1, wherein the selectively chemically cleavable site comprises a modified sugar.

7. The primer of claim 1, where the selectively chemically cleavable group is selected from the group consisting of dialkoxysilane, 3′

-(S)-phosphorothioate, 5′

-(S)-phosphorothioate, 3′

-(N)-phosphoramidate, 5′

-(N)-phosphoramidate, uracil, and ribose.

8. The primer of claim 7, wherein the selectively chemically cleavable group is 3′

-(S)-phosphorothioate or 5′

-(S)-phosphorothioate.

9. The primer of claim 1, wherein the enzyme is a DNA polymerase.

10. The primer of claim 1, wherein the enzyme is a ligase.

11. The primer of claim 1, further comprising a solid support attached to the immobilization attachment site.

12. The primer of claim 11, wherein the immobilization attachment site is attached to an intervening spacer arm bound to the solid support.

13. The primer of claim 12, wherein the intervening spacer arm is six or more atoms in length.

14. The primer of claim 11, wherein the solid support is selected from the group consisting of glass, silicon, polystyrene, aluminum, steel, iron, copper, nickel, silver and gold.

15. The primer of claim 11, wherein the solid support comprises a functionality selected from the group of avidin and streptavidin.

16. The primer of claim 11, wherein the solid support comprises an antibody.

17. The primer of claim 16, wherein the antibody comprises anti-digoxigenin.

18. The primer of claim 1, wherein the immobilization attachment site is a substituent on one of the bases or sugars of the primer.

19. The primer of claim 1, wherein the immobilization attachment site is biotin or digoxigenin.

20. The primer of claim 1, wherein the immobilization attachment site comprises a single-stranded nucleic acid.

21. The primer of claim 20, further comprising a solid support, wherein the single stranded nucleic acid is complementary to an intermediary oligonucleotide bound to the solid support and wherein the primer is attached to the solid support by hybridization of the immobilization attachment site to the intermediary oligonucleotide.

22. The primer of claim 5, wherein the chemically cleavable group incorporated into the phosphate backbone is selected from the group consisting of dialkoxysilane;

β

-cyano ether;

5′

-deoxy-5′

-aminocarbamate;

3′

-deoxy-3′

-aminocarbamate;

urea;

2′

-cyano-3′

,5′

-phospho-diester;

3′

-(S)-phosphorothioate;

5′

-(S)-phosphorothioate;

3′

-(N)-phosphoramidate;

5′

-(N)-phosphoramidate;

α

-amino amide;

vicinal diol;

ribonucleoside;

2′

-amino-3′

,5′

-phosphodiester;

allylic sulfoxide;

ester;

silyl ether;

dithioacetal;

5′

-thio-formal;

α

-hydroxy-methyl-phosphonic bisamide;

acetal; and

3′

-thio-formal.