Reciprocal subtraction differential display

• US 5,882,874 A
• Filed: 02/27/1998
• Issued: 03/16/1999
• Est. Priority Date: 02/27/1998
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Fisher, Paul B.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/032,684
• Time in Patent Office: 382 Days
• Field of Search: 435/6, 435/172.3, 435/91.2, 435/91.21
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

This invention provides a method for identifying differentially expressed nucleic acids between two samples, comprising: a) selecting a first and second nucleic acid sample; b) producing libraries for the first and second nucleic acid sample; c) performing reciprocal subtraction between the libraries to produce two subtracted libraries; d) amplifying the two subtracted libraries; and e) comparing the two amplified subtracted libraries to identify differentially expressed nucleic acids. Also, this invention provides the above-described method, wherein the 3'"'"' primer used in the PCR amplification is an oligo dT 3'"'"' primer. This invention also provides the above-described methods, wherein the comparing of step e comprises using a gel to separate the nucleic acids from both of the libraries. This invention provides the isolated nucleic acid identified by the the above-described methods, wherein the nucleic was not previously known to be differentially expressed between the two samples.

Claims

1. A method for identifying differentially expressed nucleic acids between two samples, comprising:

a. selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids;

b. performing reciprocal subtraction between the nucleic acid samples to produce two subtracted nucleic acid samples;

c. amplifying the two subtracted nucleic acid samples; and

d. comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.

4. The method of claim 1 or 2, wherein the each of the nucleic acid samples comprises a library of nucleic acids.

5. The method of claim 1 or 2, wherein the nucleic acid samples are mRNA or cDNA derived from mRNA.

6. The method of claim 1 or 2, wherein the nucleic acid samples are obtained from total RNA from E11 and E11-NMT cells.

7. The method of claim 1 or 2, wherein the first and second nucleic acid samples are obtained from cells that differ in their exposure to external factors or in their gene expression.

8. The method of claim 1 or 2, wherein the first and second nucleic acid samples are obtained from cells in different developmental stages.

9. The method of claim 1 or 2, wherein the amplifying of step (d) comprises PCR amplification.

10. The method of claim 9, wherein the PCR amplification uses a set of random primers.

11. The method of claim 9, wherein the 3'"'"' primer used in the PCR amplification is a single anchor oligo dT 3'"'"' primer.

12. The method of claim 9, wherein the 5'"'"' primer is an arbitrary primer.

13. The method of claim 1 or 2, wherein the comparing of step (e) comprises using a gel to separate the nucleic acids from both of the libraries.

14. The method of claim 1 or 2, further comprising PCR amplifying the first and second nucleic acid samples.

15. The method of claim 1 or 2, further comprising reamplifying differentially expressed nucleic acids.

16. The method of claim 1 or 2, wherein the comparing of step (e) comprises comparing the quantities of the two amplified differentially expressed nucleic acids.

17. The method of claim 1 or 2, wherein differences in the quantities of nucleic acid between the two subtracted libraries are electronically quantified.

18. The method of claim 1 or 2, wherein the libraries of step (b) are constructed with λ

-ZAP cDNA library kits.

2. A method for identifying differentially expressed nucleic acids between two samples, comprising:

a. selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids;

b. amplifying the two nucleic acid samples;

c. performing reciprocal subtraction between the amplified nucleic acid samples to produce two subtracted nucleic acid samples; and

d. comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.

3. The method of claim 2, wherein the two subtracted nucleic acid samples from step c are amplified prior to the comparing of step d.