Exponential amplification of sub-picogram nucleic acid samples with retention of quantitative representation

• US 20030211528A1
• Filed: 03/12/2003
• Published: 11/13/2003
• Est. Priority Date: 03/12/2002
• Status: Abandoned Application

• Current Assignee: Norman Iscove
• Original Assignee: Norman Iscove
• Inventors: Iscove, Norman
• Application Number:
  US10/386,103
• Publication Number:
  US 20030211528A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Within the near future it will be possible to survey expression of all genes in a sample by microarray analysis. Current methods for nucleic acid amplification require microgram amounts of complementary DNA or RNA for hybridization to microarrays. Without amplification, such amounts are only obtainable from millions of cells. However, frequently such numbers are not available: aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells are examples where few or even only single cells containing the desired information may be at hand. The current invention reduces the input amount of RNA needed for microarray analysis by a million-fold, and yields reproducible results from the picogram range of total RNA obtainable from a single cell. Of central importance to the present claims, the invention generates an amplified cDNA product in which the abundance relationships of the original RNA are faithfully preserved throughout amplification.

Claims

1. A process for generating nucleic acid targets or probes comprising the steps of:

(A) providing an RNA preparation that comprises polyadenylated mRNA;

(B) providing a first oligonucleotide primer that comprises

(1) a first segment containing a unique sequence; and

(2) a second segment being substantially complementary to the polyadenylated mRNA and capable of template-dependent first strand synthesis;

(C) contacting the mRNA with the first primer to generate by DNA polymerase or reverse transcriptase reaction from the polyadenylated mRNA, DNA strands that are substantially complementary to the polyadenylated mRNA;

(D) adding a polynucleotide tail to the 3′

end of the DNA strands whereby the DNA strands have a first portion that is complementary to the polyadenylated mRNA and a tail portion;

(E) providing a second oligonucleotide primer that comprises

(1) a first segment containing a unique sequence; and

(2) a second segment being substantially complementary to the tail portion of the DNA strand and capable of Template-dependent second strand synthesis;

(F) contacting the complementary DNA with the second primer to generate by a DNA polymerase reaction from the tailed DNA, DNA strands that are substantially complementary to the tailed DNA; and

(G) contacting the DNA strands with the first primer and the second primer to amplify exponentially, by at least 1000-fold, the DNA strands by repetitive cycles of thermal denaturation, annealing and DNA polymerase reaction, to produce the targets or probes.

2. The process of claim 1, wherein the RNA is isolated from a biological sample selected from the group consisting of a body fluid, stool a single cell, dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, cells recovered from body fluids or from the body in aspirates or scrapings or washings, a cell sorted population and a cell culture.

3. The process of claim 1, wherein the RNA is isolated from a cell or tissue selected from the group consisting of brain, liver, heart, kidney, lung, spleen, eye, retina, bone, lymph node, endocrine, endocrine gland, secretory gland, reproductive organ, blood, marrow, bone, cartilage, muscle, fat, connective tissue, nerve, vascular tissue, skin, hair, epithelial and mesothelial structures or surfaces

4. The process of claim 1 wherein the RNA is isolated from a cell or tissue selected from the group consisting of non-embryonic cell or tissue, embryonic, pathological and tumorigenic.

5. The process of claim 1, wherein the amount of RNA present is less than 10 ng.

6. The process of claim 1, wherein the polynucleotide tail is selected from the group consisting of poly(A), poly(G), poly(C) or poly(T).

7. The process of claim 1, wherein the first and second oligonucleotide primers are identical.

8. The process of claim 1, wherein the first and second oligonucleotide primers are different.

9. The process of claim 1, wherein the first and second oligonucleotide primers provide for non-directional amplification of the polyadenylated mRNA.

10. The process of claim 1, wherein the first and second oligonucleotide primers are anchored primers.

11. The process of claim 1, wherein the first and second oligonucleotide primers provide for directional amplification of the polyadenylated mRNA.

12. The process of claim 1, wherein the gene expression monitoring system is selected from the group comprising DNA array, biochip, DNA chip, DNA microarray, gene array, real time quantitative PCR.

13. The process of claim 1, further comprising digesting any residual RNA remaining after step C by the addition of RNAse H.

14. The process of claim 1, wherein the initial reverse transcription reaction occurs between about 30°

C. to about 100°

C. and the DNA polymerase reaction occurs between about 23°

C. to about 100°

C.

15. The process of claim 1, wherein the amplification comprises at least 20 cycles of denaturation, annealing, and DNA polymerase reaction.

16. The process of claim 1, wherein the amplification cycles occurs between 23°

C. to 100°

C., denaturation occurs between 90°

C. to 100°

C., annealing occurs between 37°

C. to about 75°

C., and DNA polymerase reaction occurs between about 37°

C. to about 80°

C.

17. The processes of claim 1, wherein one or more nucleotides that are covalently coupled to fluorochromes are incorporated during the repetitive cycles of thermal denaturation, annealing and DNA polymerase reaction to directly generate fluorochrome-coupled nucleic acid targets or probes.

18. The process of claim 1, wherein one or more nucleotides containing reactive side groups are incorporated during the repetitive cycles of thermal denaturation, annealing and DNA polymerase reaction, to directly generate reactive-side-group-coupled nucleic acid target or probes.

19. The process of claim 18 wherein the reactive-side-group-coupled nucleic acid targets or probes are modified by the addition of fluorochrome.

20. The method of claim 1, wherein the polyadenylated mRNA comprises between 0.1 picograms and 10 ng of RNA.

21. The method of claim 1, wherein the polyadenylated mRNA is obtained from a single cell

22. The method of claim 1, wherein the 200 to 600 nucleotides at the 3′

terminus of the mRNA are amplified.

23. A nucleic acid target produced by the process of claim 1.

24. A nucleic acid probe produced by the process of claim 1.

25. The process of claim 1, wherein the process amplifies targets to generate cDNA libraries and the representation of particular gene transcripts is measured.

26. A kit for generating nucleic acid probes for use in gene expression monitoring systems, wherein the kit comprises a reverse transcriptase, a DNA polymerase, a terminal deoxynucleotidyl transferase and oligonucleotide primers

27. A kit for generating nucleic acid targets for use in gene expression monitoring systems, wherein the kit comprises a reverse transcriptase, a DNA polymerase and oligonucleotide primers.