Method for making full-length coding sequence cDNA libraries

• US 20050175993A1
• Filed: 04/12/2002
• Published: 08/11/2005
• Est. Priority Date: 04/12/2002
• Status: Abandoned Application

• Current Assignee: Large Scale Biology Corporation
• Original Assignee: Large Scale Biology Corporation
• Inventors: Wei, Chia-Lin
• Application Number:
  US10/121,641
• Publication Number:
  US 20050175993A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

The present invention relates to a method for making cDNA libraries wherein the cDNA inserts comprise the full-length of the coding sequences but having lengths less than the full-length of the mRNA. The method comprises binding a tag molecule to a diol structure present in the 5′ Cap sites of mRNAs, forming RNA-DNA hybrids by reverse transcription to synthesize the first cDNA strand, separating RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNAs from RNA-DNA hybrids formed above by using a function of the tag molecule, and synthesizing the second cDNA strand by self-priming the first cDNA strand. The resulting cDNA libraries do not contain the full-length of the mRNAs but do contain the full-length of the coding sequences of the mRNAs.

Claims

1. A method for making a full-length coding sequence cDNA library, comprising:

(a) forming RNA-DNA hybrids by reverse transcription starting from primers using mRNAs as templates;

(b) binding a tag molecule to a diol structure present in the 5′

cap site of a mRNA forming a RNA-DNA hybrid; and

(c) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA from the RNA-DNA hybrids formed above by binding the tag molecule, wherein said DNA corresponding to a full-length mRNA are first cDNA strands;

wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs.

2. The method according to claim 1, further comprising the step of synthesizing second cDNA strands using as templates said first cDNA strands, wherein ligating an RNA or DNA oligomer to the 3′

end of said first cDNA strands is not required.

3. The method according to claim 2, said synthesizing comprises self-priming said first cDNA strand.

4. The method according to claim 1, further comprising the step of digesting RNA-DNA hybrids binding tag molecules with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the RNA-DNA hybrids carrying a DNA not corresponding to a full-length mRNA to remove tag molecules from the hybrids.

5. The method according to claim 4, wherein said synthesizing and said digesting at least overlap.

6. The method according to claim 4, wherein said RNase capable of cleaving single stranded RNA is ribonuclease I.

7. The method according to claim 1, wherein the primer is oligo dT.

8. The method according to claim 1, wherein the diol structure present in the 5′

cap site of the mRNA is subjected to a ring-open reaction by oxidation with periodic acid to form a dialdehyde and the dialdehyde is reacted with a tag molecule having a hydrazine terminus to form a mRNA binding the tag molecule.

9. The method according to claim 8, wherein the tag molecule having a hydrazine terminus is a biotin molecule having a hydrazine terminus or an avidin or streptavidin molecule having hydrazine terminus.

10. The method according to claim 1, wherein the tag molecule is a biotin molecule having a functional group which is capable of binding a diol structure present in the 5′

cap site of mRNA, and the hybrids carrying a DNA corresponding to a full-length mRNA are separated by utilizing binding properties of avidin or streptavidin fixed on a solid support to the biotin molecule which is the tag molecule of the RNA-DNA hybrid.

11. The method according to claim 1, wherein the tag molecule is an avidin or streptavidin molecule having a functional group which is capable of binding a diol structure present in the 5′

cap site of mRNA, and the hybrids carrying a DNA corresponding to a full-length mRNA are separated by utilizing binding properties of biotin fixed on a solid support to the avidin or streptavidin molecule which is the tag molecule of the RNA-DNA hybrid.

12. A method for constructing a full-length coding sequence cDNA library, comprising:

(a) binding a tag molecule to a diol structure present in 5′

cap sites of mRNAs by oxidizing the 5′

cap site diol to form a dialdehyde and reacting the resulting dialdehyde with a tag molecule having a group reactive with the dialdehyde;

(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs binding the tag molecule as templates; and

(c) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNA from the RNA-DNA hybrids formed above by using a function of the tag molecule, wherein said DNA corresponding to a full-length mRNA are first cDNA strands;

wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs.

13. The method according to claim 12, further comprising the step of synthesizing second cDNA strands using as templates said first cDNA strands, wherein ligating an RNA or DNA oligomer to the 3′

end of said first cDNA strands is not required.

14. The method according to claim 13, said synthesizing comprises self-priming said first cDNA strand.

15. The method according to claim 12, further comprising the step of digesting RNA-DNA hybrids binding tag molecules with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the RNA-DNA hybrids carrying a DNA not corresponding to a full-length mRNA to remove tag molecules from the hybrids.

16. The method according to claim 15, wherein said synthesizing and said digesting at least overlap.

17. The method according to claim 12, wherein the primer is oligo dT.

18. The method according to claim 12, wherein the tag molecule is a biotin molecule having a functional group capable of binding to a diol structure present in 5′

cap site of mRNA and the RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNAs are separated by utilizing binding between an avidin molecule fixed on a solid support and a biotin molecule possessed by the RNA-DNA hybrids as the tag molecule.

19. The method according to claim 12, wherein the tag molecule is an avidin molecule having a functional group capable of binding to a diol structure present in 5′

cap site of mRNA and the RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNAs are separated by utilizing binding between a biotin molecule fixed on a solid support and an avidin molecule possessed by the RNA-DNA hybrids as the tag molecule.

20. The method according to claim 12, wherein the diol structure present in 5′

Cap site of mRNA is subjected to a ring-open reaction by oxidation with sodium periodate to form a dialdehyde and the dialdehyde is reacted with a tag molecule having a hydrazine terminus to form mRNA binding the tag molecule.

21. The method according to claim 20, wherein the tag molecule having a hydrazine terminus is a biotin molecule or avidin molecule having a hydrazine terminus.

22. The method according to claim 12, wherein the RNA-DNA hybrids are digested with an RNase capable of cleaving single strand RNA to cleave the single strand parts of the hybrids so that the tag molecule is removed from those hybrids carrying a DNA not corresponding to a full-length mRNAs and then those hybrids carrying a tag molecule and a DNA corresponding to a full-length of mRNAs are separated.

23. The method according to claim 22, wherein the RNase capable of cleaving single strand RNA is ribonuclease I.

24. A method for making a full-length cDNA library, comprising:

(a) binding a biotin molecule to a diol structure present in 5′

cap sites of mRNAs by oxidizing the 5′

cap site diol to form a dialdehyde and reacting the resulting dialdehyde with a biotin molecule having a group reactive with the dialdehyde;

(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs bound to biotin molecules as templates;

(c) digesting the RNA-DNA hybrids with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the hybrids carrying a DNA not corresponding to a full-length mRNA to remove biotin molecules from the hybrids, wherein said DNA not corresponding to a full-length mRNA is a first cDNA strand; and

(d) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA and binding the biotin molecules by (1) allowing them to react with avidin fixed on a solid support or (2) affinity chromatography to a solid support;

wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs.

25. The method according to claim 24, further comprising the step of synthesizing second cDNA strands using as templates said first cDNA strands, wherein ligating an RNA or DNA oligomer to the 3′

end of said first cDNA strands is not required.

26. The method according to claim 25, said synthesizing comprises self-priming said first cDNA strand.

27. The method according to claim 26, wherein said synthesizing and said digesting at least overlap.

28. The method according to claim 27, wherein the primer is oligo dT and the RNase capable of cleaving single strand RNA is ribonuclease I.

29. A method for making a fall-length cDNA library, comprising:

(a) binding an avidin molecule to a diol structure present in 5′

cap sites of mRNAs by oxidizing the 5′

cap site diol to form a dialdehyde and reacting the resulting dialdehyde with an avidin molecule having a group reactive with the dialdehyde;

(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs bound to avidin molecules as templates;

(c) digesting the RNA-DNA hybrids with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the hybrids carrying a DNA not corresponding to full-length mRNAs to remove avidin molecules from the hybrids, wherein said DNA not corresponding to a full-length mRNA is a first cDNA strand; and

(d) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA and binding avidin molecules by (1) allowing them to react with biotin fixed on a solid support or (2) affinity chromatography to a solid support;

wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs.

30. The method according to claim 29, further comprising the step of synthesizing second cDNA strands using as templates said first cDNA strands, wherein ligating an RNA or DNA oligomer to the 3′

end of said first cDNA strands is not required.

31. The method according to claim 30, said synthesizing comprises self-priming said first cDNA strand.

32. The method according to claim 31, wherein said synthesizing and said digesting at least overlap.

33. The method according to claim 32, wherein the primer is oligo dT and the RNase capable of cleaving single strand RNA is ribonuclease I.