Enzyme digestion method for the detection of amplified DNA

• US 5,580,730 A
• Filed: 08/19/1994
• Issued: 12/03/1996
• Est. Priority Date: 08/19/1994
• Status: Expired due to Fees

• Current Assignee: Olympus America Incorporated (Olympus Corporation)
• Original Assignee: Olympus America Incorporated (Olympus Corporation)
• Inventors: Okamoto, Naoaki
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/293,638
• Time in Patent Office: 837 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/15, 435/18, 536/26.6, 536/24.33, 935/77, 935/78
• US Class Current: 435/6.12
• 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 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Qualitative and quantitative methods for detecting the presence of double-stranded, non-5'"'"'-phosphorylated DNA in samples that may also contain 5'"'"'-phosphorylated DNA and/or single-stranded DNA are described. These methods involve treating the sample with an enzyme that specifically degrades 5'"'"'-phosphorylated DNA together with an enzyme that specifically degrades single-stranded DNA. More specifically, methods are described for the detection of the products of DNA amplification reactions, such as the polymerase chain reaction (PCR), wherein post-amplification enzyme digestion substantially reduces or eliminates background signals that are apparently caused by the presence of template DNA and primers in the sample after amplification is complete.

Claims

1. A method for detecting the presence of double-stranded, non-5'"'"'-phosphorylated DNA in a sample that may also contain 5'"'"'-phosphorylated DNA and single-stranded DNA, comprising:

(a) adding to the sample an enzyme that specifically degrades single stranded DNA and an enzyme that specifically degrades DNA having 5'"'"'-phosphorylated ends, and incubating under conditions that permit the activity of said enzymes;

(b) detecting the presence of DNA in the sample of step (a) wherein includes said DNA double stranded, non-5'"'"'-phosphorylated DNA.

2. A method for detecting the presence of one or more target DNA sequences in a sample containing 5'"'"'-phosphorylated DNA, comprising:

(a) carrying out enzymatic amplification of one or more portions of the DNA in said sample to produce one or more amplification products wherein at least a portion of each of said amplification products is double stranded, non-5'"'"'-phosphorylated DNA;

(b) adding an enzyme that specifically degrades single stranded DNA and an enzyme that specifically degrades DNA having 5'"'"'-phosphorylated ends, and incubating under conditions that permit the activity of said enzymes;

(c) detecting the presence of DNA in the sample of step (b) wherein said DNA includes double stranded, non-5'"'"'-phosphorylated DNA.

3. The method of claim 2 wherein said 5'"'"'-phosphorylated DNA is biologically-derived.

4. The method of claim 2 wherein each of said steps are carried out in succession by addition of reagents to the reaction mixture resulting from the previous step, without any intervening purifications or separations.

5. The method of claim 2 wherein all steps are carried out in the same sample vessel.

6. The method of claim 5 wherein said sample vessel is a well of a microtiter plate.

7. The method of claim 2 wherein said detection of DNA is quantitative.

8. The method of claim 2 wherein said detection is qualitative, the presence or absence of amplified DNA in an unknown sample being determined by comparison with the results obtained using one or more known samples.

9. The method of claim 2 wherein said step of detecting the presence of DNA in the sample further comprises adding an intercalating fluorophore to said sample and detecting the fluorescence of the DNA when the resulting sample is excited by an appropriate excitation beam.

10. The method of claim 9 wherein said intercalating fluorophore is selected from the group of ethidium bromide (EtBr), ethidium homodimer (EthD), Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and thiazole orange monomer (TO-PRO).

11. A method for detecting the presence of one or more target DNA sequences in a sample containing 5'"'"'-phosphorylated DNA, comprising:

(a) carrying out primed enzymatic transcription on one or more portions of the DNA in said sample, using one or more primers that do not have 5'"'"'-phosphorylated ends;

(b) adding to the DNA of said sample an enzyme that specifically degrades single stranded DNA, and incubating under conditions that permit the activity of said enzyme;

(c) adding to the DNA remaining after step (b) an enzyme that specifically degrades DNA having 5'"'"'-phosphorylated ends, and incubating under conditions that permit the activity of said enzyme;

(d) detecting the presence of DNA in the sample of step (c) wherein said DNA includes double stranded, non-5'"'"'-phosphorylated DNA.

12. A method for detecting the presence of one or more target DNA sequences in a sample containing 5'"'"'-phosphorylated DNA, comprising:

(a) amplifying one or more portions of the DNA in said sample by polymerase chain reaction (PCR) to produce one or more double-stranded, non-5'"'"'-phosphorylated amplification products;

(b) adding the enzymes lambda exonuclease and Exonuclease I and incubating under conditions that permit the activity of said enzymes;

(c) adding an intercalating fluorophore selected from the group consisting of ethidium bromide (EtBr), ethidium homodimer (EthD), Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and thiazole orange monomer (TO-PRO);

(d) detecting the level of fluorescence emitted from the DNA in the sample of step (c) when illuminated with an appropriate excitation beam wherein said DNA includes double stranded, non-5'"'"'-phosphorylated DNA.

13. The method of claim 12 wherein said level of fluorescence is compared with the fluorescence levels observed when one or more known samples were analyzed by the method of claim 13.

14. The method of claim 12 wherein said PCR amplification is carried out using sequence specific primers.

15. The method of claim 14 wherein said sequence specific primers are specific for one or more human leukocyte antigens (HLAs).

16. A homogeneous method for detecting the presence or absence of one or more human leukocyte antigen (HLA) in a sample that is substantially of human biological origin, comprising:

(a) using one or more HLA-sequence specific antigens in a polymerase chain reaction (PCR) to amplify one or more portions of the HLA-specific DNA in said sample;

(b) adding the enzymes lambda exonuclease and Exonuclease I and incubating under conditions that permit the activity of said enzymes;

(c) adding an intercalating fluorophore selected from the group of ethidium bromide (EtBr), ethidium homodimer (EthD), Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and thiazole orange monomer (TO-PRO);

(d) detecting the level of fluorescence emitted from the DNA in the sample of step (c) when illuminated with an appropriate excitation beam wherein said DNA includes double stranded, non-5'"'"'-phosphorylated DNA.