Nucleic acid amplification methods

• US 6,593,086 B2
• Filed: 12/01/2000
• Issued: 07/15/2003
• Est. Priority Date: 05/20/1996
• Status: Expired due to Term

• Current Assignee: Icahn School of Medicine at Mount Sinai (The Mount Sinai Health System, Inc.)
• Original Assignee: Icahn School of Medicine at Mount Sinai (The Mount Sinai Health System, Inc.)
• Inventors: Zhang, David Y.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/728,265
• Publication Number:
  US 20020182598A1
• Time in Patent Office: 956 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/68, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 424/184.1, 424/193.1, 424/194.1, 424/130.1
• US Class Current: 435/6.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/137 a displacement step
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to assays and kits for carrying out said assays for the rapid, automated detection of infectious pathogenic agents and normal and abnormal genes. The present invention further relates to methods for general amplification of genomic DNA and total mRNAs and for analyzing differential mRNA expression using the amplification methods disclosed herein.

Claims

1. A method for detection of a target nucleic acid within a cell comprising the steps of:

(a) embedding the cell to be analyzed for the presence of the target nucleic acid within a matrix;

(b) adding a circularizable Amp-probe-2 having 3′ and

5′

regions that are complementary to adjacent but noncontiguous sequences in the target nucleic acid, said 3′ and

5′

regions separated by a linker region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid such that a complex is formed comprising the target nucleic acid and Amp-probe-2;

(c) ligating the 3′ and

5′

ends of said circularizable Amp-probe-2 with a ligating agent that joins nucleotide sequences such that a circular Amp-probe-2 is formed;

(d) washing said complex; and

(e) detecting said complex, wherein the detection thereof indicates the presence of the target nucleic acid in the embedded cell.

2. The method of claim 1, wherein said linker region is labeled with a ligand, such that a complex is formed comprising the target nucleic acid and the circular Amp-probe-2.

3. The method of claim 2, wherein a ligand binding moiety is added which binds to and forms an affinity pair with said ligand and an oligonucleotide signal probe internally labeled with said ligand whereby said ligand binding moiety binds to said ligand on said Amp-probe-2 and also to said ligand on said oligonucleotide signal probe thereby forming a labeled complex.

4. The method of claim 2, wherein said ligand is selected from the group consisting of biotin, antigens, haptens, antibodies, heavy metal derivatives, and polynucleotides.

5. The method of claim 3 wherein said ligand binding moiety is selected from the group consisting of streptavidin, avidin, antibodies, antigens, thio groups and polynucleotides.

6. The method of claim 1, wherein said cells are obtained from clinical samples obtained for testing purposes.

7. The method of claim 6, wherein said sample is selected from the group consisting of whole blood, separated white blood cells, sputum, urine, tissue biopsies and throat swabbings.

8. The method of claim 1, wherein said matrix is selected from the group consisting of polyacrylamide and agarose.

9. The method of claim 1, wherein a DNA polymerase is added to extend the region between said 5′

and 3′

regions.

10. The method of claim 1, wherein said circular Amp-probe-2 is amplified by contacting said complex with one or more forward RAM primers that are complementary and hybridizable to a portion of the linker region of the Amp-probe-2 and dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the forward RAM primers are extended around the circle for multiple revolutions to form a single-stranded DNA of repeating units complementary to the sequence of the circular probe.

11. The method of claim 10, wherein said single-stranded DNA is detected and said detection indicates the presence of the target nucleic acid in the embedded cell.

12. The method of claim 1, wherein said circular Amp-probe-2 is amplified by contacting said complex with one or more forward RAM primers that are complementary and hybridizable to a portion of the linker region of the Amp-probe-2 and one or more reverse RAM primers that are substantially identical to a portion of the linker region of the Amp-probe-2 that does not overlap with the portion of the linker region to which the toward Ram Primer is complementary, dNTPS and a DNA polymerase having strand displacement activity, under conditions where the forward RAM primers are extended around the circle for multiple revolutions to form a single-stranded DNA of repeating units complementary to the sequence of the circular probe, and multiple copies of the reverse RAM primer hybridize to complementary regions of the single-stranded DNA and are extended by the DNA polymerase to provide extension products, and whereby the extension products of the reverse RAM primers displace downstream copies of the reverse RAM primers and corresponding extension products to provide displaced single strands to which multiple copies of said forward RAM primer bind and are extended by the DNA polymerase.

13. The method of claim 12, wherein said amplified DNA is detected and said detection indicates the presence of the target nucleic acid in the embedded cell.