Method for reducing carryover contamination in an amplification procedure

• US 5,427,929 A
• Filed: 05/03/1993
• Issued: 06/27/1995
• Est. Priority Date: 05/01/1990
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Richards, Rodney M., Jones, Theodore, Snitman, David L., Brown, Gregory S.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/057,192
• Time in Patent Office: 785 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention provides an efficient and economical method for reducing carryover contamination in an amplification procedure. The method of the present invention enables background caused by contaminant amplification product to be reduced or eliminated through the incorporation of at least one modification into the amplification product. The modified amplification product is readily distinguishable from the target sequence in a test sample. Prior to amplifying the target in a new test sample, the sample may be treated to selectively cleave the contaminant amplification product so that it cannot be amplified in the new sample.

Claims

1. A method for reducing DNA carryover contamination in a first test sample which may contain amplification product from a previously amplified DNA target-containing test sample comprising:

(a) amplifying said DNA target of said previous test sample in the presence of at least one modified amplification probe or primer to generate a modified amplification product having an enzyme recognition site or chemically cleavable site not present in said target; and

,

(b) contacting said first test sample with a means for cleaving said modified amplification product to reduce the amount of modified amplification product from said previous test sample thereby reducing DNA carryover contamination present in said first test sample.

2. The method of claim 1 wherein said modified amplification product has is an enzyme recognition site and said means for cleaving said modified amplification product is an enzyme.

3. The method of claim 2 wherein said enzyme recognition site is an RNAse recognition site and said means for cleaving said modified amplification product is an RNAse.

4. The method of claim 3 wherein said RNAse is selected from the group consisting of RNAse H and RNAse A.

5. The method of claim 4 wherein said RNAse is RNAse A.

6. The method of claim 2 wherein said enzyme recognition site is a restriction enzyme recognition site and said means for cleaving said modified amplification product is a restriction enzyme.

7. The method of claim 6 wherein said restriction enzyme recognition site is recognized by a remote cutting restriction enzyme.

8. The method of claim 7 wherein said remote cutting restriction enzyme is Fok I.

9. The method of claim 1 wherein said modified amplification product has a chemically cleavable site.

10. The method of claim 9 wherein said chemically cleavable site is incorporated into an amplification probe by joining the 3'"'"'- and 5'"'"'-ends of partial probe sequences with a homobifunctional linking reagent selected from the group consisting of dithiobis(succinimidyl-propionate), disuccinimidyl-tartarate, and ethylene glycolbis (succinimidylsuccinate).

11. The method of claim 9 wherein said chemically cleavable site comprises an RNA base substitution.

12. A kit for reducing DNA carryover contamination in a polymerase chain reaction type of amplification procedure comprising:

(a) at least one modified amplification primer which is capable of incorporating at least one modification into a PCR-derived amplification product such that the resulting modified PCR-derived amplification product has a remote cutting restriction enzyme recognition site not present in said target sequence; and

,

(b) a remote cutting restriction enzyme.

13. The kit of claim 12 wherein said remote cutting restriction enzyme is Fok I.

14. A kit for reducing DNA carryover contamination in a ligase chain reaction type of amplification procedure comprising:

(a) at least one upper strand and at least one lower strand modified amplification probe which are capable of incorporating at least one upper strand and at least one lower strand modification into a remote cutting restriction LCR-derived amplification product such that the resulting modified LCR-derived amplification product has an enzyme recognition site not present in said target sequence; and

,

(b) a remote cutting restriction enzyme.

15. The kit of claim 14 wherein said remote cutting restriction enzyme is Fok I.

16. A method for reducing DNA carryover contamination in a method for amplifying a target sequence comprising the steps of:

(a) hybridizing at least two amplification primers to said target sequence in the presence of an excess of deoxynuclcoside triphosphates and a polymerizing agent, wherein at least one of said amplification primers is a modified amplification primer having a modification selected from the group consisting of an enzyme recognition site and a chemically cleavable site;

(b) extending said amplification primers with said deoxynucleoside triphosphates and said polymerizing agent to generate amplification product such that modified amplification product is generated from extension of each modified amplification primer;

(c) separating said modified amplification product from said target sequence;

(d) repeating steps (a) through (c); and

(e) cleaving said modified amplification product thereby reducing DNA carryover contamination in a method for amplifying a target sequence.

17. The method of claim 16 wherein step (e) is performed at an beginning of the amplification procedure.

18. The method of claim 16 further comprising:

(f) detecting the amount of cleaved modified amplification product after performing g step (e).

19. A method for reducing DNA carryover contamination in a method for amplifying a denatured double-stranded target sequence comprising the steps of:

(a) contiguously hybridizing at least two pairs of amplification probes to said denatured double stranded target sequence, wherein at least one upper strand and at least one lower strand of said pairs of amplification probes is a modified amplification probe having a modification selected from the group consisting of an enzyme recognition site and a chemically clearable site;

(b) ligating said contiguously hybridized amplification probes to generate modified amplification product thereby reducing DNA carryover contamination in a method for amplifying a denatured double stranded target sequence;

(c) separating said modified amplification product from said target sequence;

(d) repeating steps (a) through (c);

(e) cleaving said modified amplification product.

20. The method of claim 19 wherein step (e) is performed at a beginning of the amplification procedure.

21. The method of claim 19 further comprising:

(f) detecting the amount of cleaved modified amplification product after performing step (e).