Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample

• US 5,795,722 A
• Filed: 03/18/1997
• Issued: 08/18/1998
• Est. Priority Date: 03/18/1997
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Visible Genetics, Inc. (Bayer AG)
• Inventors: Dunn, James M., Lacroix, Jean-Michel
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/819,912
• Time in Patent Office: 518 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS

Abstract

A method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte first combines the sample with a control nucleic acid, and two primer pairs, a first primer pair effective to amplify a conserved region of the nucleic acid analyte if present in the sample to produce a conserved fragment having a first length and to amplify the control nucleic acid to produce a control fragment having a second length different from the first length, and a second primer pair effective to amplify a second region of the nucleic acid analyte to produce a sequencing fragment. One member of the first primer pair is labeled with a detectable label, and one member of the second primer pair may be labeled with a label such as biotin effective to permit capture of the primer. The combined mixture is then processed to amplify the sample and control nucleic acid using the first and second primer pairs to produce an amplification product mixture containing conserved fragments, sequencing fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragment when the nucleic acid analyte is not present in the sample. This product mixture is analyzed to determine the relative amounts of conserved fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample and used as a starting point for a reaction to determine the sequence of the sequencing fragment.

Claims

1. A method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte, comprising the steps of:

(a) combining the sample with a control nucleic acid, and two primer pairs,

a first primer pair effective to amplify a conserved region of the nucleic acid analyte if present in the sample to produce a conserved fragment having a first length and to amplify the control nucleic acid to produce a control fragment having a second length different from the first length, one member of the first primer pair being labeled with a detectable label, and

a second primer pair effective to amplify a second region of the nucleic acid analyte to produce a sequencing fragment, one member of the second primer pair being labeled with a label effective to permit capture of the primer;

(b) amplifying the sample and control nucleic acid using the first and second primer pairs to produce an amplification product mixture containing conserved fragments, sequencing fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragment when the nucleic acid analyte is not present in the sample;

(c) analyzing the relative amounts of conserved fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample; and

(d) determining the sequence of the sequencing fragment in the amplification mixture to determine the qualitative characteristics of any nucleic acid analyte in the sample.

2. The method according to claim 1, wherein a plurality of control nucleic acids are added to the sample, each control nucleic acid producing a corresponding control fragment having a different length, and each control nucleic acid being added to the sample in different amounts.

3. The method according to claim 1, wherein at least a first and a second control nucleic acid are added to the sample, and the amounts of the first and second control nucleic acid differ by a factor of 10 or more.

4. The method according to claim 1, wherein the length of the conserved fragment differs from the length of the control fragment by at least 10 nucleotides.

5. The method according to claim 1, wherein the length of the conserved fragment differs from the length of the control fragment by at least 25 nucleotides.

6. The method according to claim 1, wherein the length of the conserved fragment differs from the length of the control fragment by at least 50 nucleotides.

7. The method according to claim 1, wherein the relative amounts of conserved fragments and control fragments are analyzed by separation of the fragments by gel electrophoresis and detection of the separated fragments.

8. The method according to claim 7, wherein the separation and detection of the separated fragments is performed on an instrument having a dynamic range of at least three orders of magnitude.

9. The method according to claim 7, wherein the separation and detection of the separated fragments is performed on an instrument having a dynamic range of at least three orders of magnitude.

10. The method according to claim 1, wherein the detectable label is a fluorescent label.

11. The method according to claim 1, wherein the label effective to permit capture of one member of the second primer pair is biotin.

12. The method according to claim 11, wherein the detectable label is a fluorescent label.

13. The method according to claim 12, wherein the relative amounts of conserved fragments and control fragments are analyzed by separation of the fragments by gel electrophoresis and detection of the separated fragments.

14. The method according to claim 13 wherein the separation and detection of the separated fragments is performed on an instrument having a dynamic range of at least three orders of magnitude.

15. The method according to claim 13, wherein the separation and detection of the separated fragments is performed on an instrument having a dynamic range of at least three orders of magnitude.

16. The method according to claim 1, wherein the nucleic acid analyte is HIV-1 RNA or corresponding cDNA or proviral DNA which is integrated into the genome of a host cell.

17. The method according to claim 16, wherein the first primer pair amplifies a conserved region within the gag genes of HIV-1.

18. The method according to claim 16, wherein the second primer pair amplifies a region within the protease gene of HIV-1.

19. The method according to claim 18, wherein the first primer pair amplifies a conserved region within the gag genes of HIV-1.

20. The method according to claim 1, wherein the first and second primer pairs are the same, and wherein one member of the pair has a detectable label and the other member of the pair has a label effective to permit capture of the primer.

21. The method according to claim 1, further comprising the step of capturing the sequencing fragment using the label effective to permit capture of one member of the second primer pair, thereby separating the sequencing fragment from the remainder of the amplification product mixture prior to sequencing the sequencing fragment.

22. The method according to claim 21, wherein the label effective to permit capture of one member of the second primer pair is biotin.

23. The method according to claim 1, wherein sequencing is performed on both the sense and antisense strands of the sequencing fragment using two species of distinctly labeled sequencing primers, said two species of sequencing primers each binding to a different one of the sense and antisense strands of the sequencing fragment.

24. The method according to claim 1, wherein the nucleic acid analyte is an RNA, further comprising the step of reverse transcribing the nucleic acid analyte prior to the amplification step.

25. A kit for quantitative and qualitative evaluation of a target nucleic acid analyte in a sample comprising, in packaged combination,

(a) a control nucleic acid that is structurally related to a conserved region of the target nucleic acid analyte;

(b) a first primer pair for amplification of the control nucleic and the conserved region, one member of the first primer pair being labeled with a detectable label; and

(c) a second primer pair for amplification of a second region of the nucleic acid analyte different from the first region.

26. The kit according to claim 25, wherein at least member of the second primer pair is labeled with a capture moiety to facilitate separation of the amplification product of this primer pair from a reaction mixture.

27. The kit according to claim 26, wherein the capture moiety to biotin.