Method of obtaining a library of tags capable of defining a specific state of a biological sample

• US 6,506,561 B1
• Filed: 04/29/1999
• Issued: 01/14/2003
• Est. Priority Date: 01/27/1999
• Status: Expired due to Fees

• Current Assignee: Commissariat a L'Energie Atomique (AES Corporation), Centre National De La Recherche Scientifique
• Original Assignee: Commissariat a L'Energie Atomique (AES Corporation), Centre National De La Recherche Scientifique
• Inventors: Virlon, Bérangère, Cheval, Lydie, Elalouf, Jean-Marc
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/301,721
• Time in Patent Office: 1,356 Days
• Field of Search: 435/6, 435/91.1, 536/22.1, 536/24.2, 935/1, 935/5, 935/6, 935/8, 935/9, 935/16, 935/19, 935/4
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]

Abstract

A method of obtaining a library of tags able to define a specific state of a biological sample, such as a tissue or a cell culture. The present method provides an important advantage over other methods used to analyze gene expression in that libraries may be generated from tiny amounts of cells, e.g., from 30,000-100,000 cells.

Claims

1. A method of obtained a library of tags capable of defining a specific state of a biological sample, comprising:

(1) extracting mRNA from a biological sample comprising ≦

5×

10⁶ cells, corresponding to at most 50 μ

g of total RNA or 1 μ

g of poly(A) RNA, by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,

(2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;

(a) synthesizing the 1^st strand of the cDNA by reverse transcription of the mRNA template into a 1^st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and

(b) synthesizing the 2^nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an E. coli DNA polymerase,

(3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme,

(4) separating the cleaved cDNAs in two aliquots,

(5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site two different hybrid DNA molecules composed from linkers 1A and 1B or from linkers 2A and 2B, wherein the linkers have the following formulas;

2. The method of claim 1, wherein the biological sample is a tissue sample or a cell culture.

3. The method of claim 1, wherein the DNA polymerase is a T7 DNA polymerase or a Vent polymerase.

4. The method of claim 1, wherein the oligo(dT) is an oligo(dT)₂₅.

5. The method of claim 1, wherein in step (1), after mRNA binding to beads, different washes are performed in washing buffer supplemented with glycogen.

6. The method of claim 1, wherein the two different oligonucleotide linkers further comprise amplification primer hybridization sequences, the method further comprises amplifying the ditags.

7. The method of claim 6, wherein the ditags are amplified with primers having the following formula:

8. The method of claim 1, wherein step (9) further comprises producing and cloning concatemers of the ditags.

9. The method of claim 5, wherein the concatemers of the ditags have more than 300 bp.

10. The method of claim 1, wherein the biological sample is from nephron segments and contains about 15,000 to 45,000 cells, corresponding to 0.15-0.45 μ

g of total RNA.

11. The method of claim 1, wherein in step (2) the synthesis of the 1^st strand of a cDNA is performed with Moloney Murine Leukaemia Virus reverse transcriptase (M-MLV RT), and oligo(dT)₂₅ as primers.

12. The method of claim 1, wherein the amount of each linker in step (5) is at most 8-10 pmol and comprised between 0.5 pmol and 8 pmol for initial amounts of respectively 10-40 ng of mRNAs and 5 μ

g of mRNAs.

13. A method of obtaining a library of tags able to define a specific state of biological sample, comprising:

(1) extracting mRNA from biological sample comprising ≦

5×

10⁶ cells, corresponding to at most 50 μ

g of total RNA or 1 μ

g of poly(A) RNA, by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,

(2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;

(a) synthesizing the 1^st strand of the cDNA by reverse transcription of the mRNA template into a 1^st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and

(b) synthesizing a 2^nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid-form by an E. coli DNA polymerase,

(3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme,

(4) separating the cleaved cDNAs in two aliquots,

(5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site to a linker consisting of one double-strand cDNA molecule composed from linkers 1A and 1B or from linkers 2A and 2B. wherein the linkers have the following formulas;

14. The method of claim 13, wherein the biological sample is a tissue sample or a cell culture.

15. The method of claim 13, wherein the DNA polymerase is a T7 DNA polymerase or a Vent polymerase.

16. The method of claim 13, wherein the oligo(dT) is as oligo(dT)₂₅.

17. The method of claim 13, wherein the primers of step (9) have the following formulas:

18. The method of claim 13, wherein the tissue sample is from nephron segments and contains about 15,000 to 45,000 cells, corresponding to 0.15-0.45 μ

g of total RNA.

19. The method of claim 13, wherein in step (1), after mRNA binding to beads, different washes are performed in washing buffer supplemented with glycogen.