Nucleic acid amplification methods

• US 8,632,999 B1
• Filed: 06/13/2003
• Issued: 01/21/2014
• Est. Priority Date: 06/22/1994
• Status: Expired due to Term

• Current Assignee: David Y. Zhang
• Original Assignee: David Y. Zhang
• Inventors: Zhang, David Y.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US10/461,848
• Publication Number:
  US 20140038238A1
• Time in Patent Office: 3,875 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.51
• 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 total mRNAs and for analyzing differential mRNA expression using the amplification methods disclosed herein.

Claims

1. A method for amplifying a mRNA comprising:

(a) hybridizing a Capture/Amp-probe to the 3′

poly-A sequence of a mRNA, wherein the Capture/Amp-probe contains multiple RAM primer sites, wherein the primer site is 10 to 60 nucleotides in length;

(b) reverse transcribing of the mRNA to generate a single stranded cDNA such that the cDNA contains a poly-T sequence;

(c) converting the single stranded cDNA of step (b) to double stranded DNA, wherein the single strand of DNA of the double stranded DNA of step (c) which is complementary to the single stranded cDNA of step (b) contains a poly-A sequence;

(d) ligating with a ligating agent at least one double stranded Amp-probe to one end of the double stranded DNA, whereby the Amp-probe is ligated to the end opposite the poly-A sequence in the double stranded DNA, wherein the Amp-probe contains multiple RAM primer sites;

(e) denaturing the double stranded DNA; and

(f) amplifying the DNA under isothermal conditions by contacting the DNA with;

(1) at least two RAM primers that are complementary and hybridizable to the RAM primer sites;

(2) dNTPs; and

(3) a DNA polymerase having strand displacement activity, under isothermal conditions whereby the RAM primers are extended from the RAM primer sites to the end of the DNA to form multiple single stranded DNAs.

2. The method of claim 1, wherein the DNA polymerase is φ

29 DNA polymerase or Bst DNA polymerase.

3. The method of claim 1, wherein ligation of the Amp-probe is performed in a matrix.

4. The method of claim 1, wherein the ligating agent is an enzyme or a chemical agent.

5. The method of claim 4, wherein the enzyme is a DNA ligase.

6. The method of claim 5, wherein the DNA ligase is T₄ DNA ligase or Taq DNA ligase.