Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides

• US 4,661,450 A
• Filed: 05/03/1983
• Issued: 04/28/1987
• Est. Priority Date: 05/03/1983
• Status: Expired due to Fees

• Current Assignee: Molecular Genetics Research and Development Limited Partnership
• Original Assignee: Molecular Genetics Research and Development Limited Partnership
• Inventors: Purchio, Anthony F., Kempe, Tomas, DeLorbe, William J., Collett, Marc S.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Teskin, Robin Lyn
• Application Number:
  US06/491,099
• Time in Patent Office: 1,456 Days
• Field of Search: 536/27, 536/28, 536/29, 435/172.3, 435/68, 435/70, 435/317, 435/91
• US Class Current: 435/91.41
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Methods and compositions for the insertion and molecular cloning of RNA in DNA cloning vectors are described. RNA molecules to be cloned are modified by the ligation of oligonucleotide linkers onto the termini of the RNA molecule using T4 RNA ligase. Such linkers may be composed of DNA, RNA, or mixtures of each and facilitate the insertion and ligation of the RNA species into a DNA cloning vector. The resulting recombinant vectors are used to transform host cells which provide for the generation of multiple DNA copies of the RNA molecule.

Claims

1. A method for the insertion of a single-stranded RNA sequence into a cloning vector, comprising:

annealing both termini of the single-stranded RNA sequence with a linear double-stranded cloning vector having single-stranded termini complementary to both termini of the single-stranded RNA sequence to form an annealed product, in which the complementary termini of the single-stranded RNA sequence where formed by attaching oligonucleotide linkers to the RNA sequence by means of an RNA ligase.

2. The method according to claim 1 wherein said RNA ligase is T4 RNA ligase.

3. The method according to claim 2 wherein the oligonucleotide linker attached to the 3'"'"'-terminus of the RNA sequence is composed of DNA.

4. The method according to claim 2 wherein the oligonucleotide linker attached to the 3'"'"'-terminus of the RNA sequence is composed of RNA.

5. The method according to claim 2 wherein the oligonucleotide linker attached to the 3'"'"'-terminus of the RNA sequence is composed of a mixture of DNA and RNA.

6. The method according to claim 3 wherein the DNA linker is single-stranded.

7. The method according to claim 3 wherein the DNA linker is double-stranded.

8. The method according to claim 2 wherein the oligonucleotide linker attached to the 5'"'"'-terminus of the RNA sequence is composed of a mixture of DNA and RNA such that the 3'"'"'-terminus of the oligonucleotide linker is a ribonucleotide.

9. The method according to claim 2 wherein the oligonucleotide linker attached to the 5'"'"'-terminus of the RNA sequence is composed of RNA.

10. The method according to claim 8 wherein the oligonucleotide linker is single-stranded.

11. The method according to claim 8 wherein the oligonucleotide linker is double-stranded.

12. The method according to claim 1 wherein said oligonucleotide linkers contain a DNA sequence encoding a restriction endonuclease recognition site.

13. The method according to claim 12 wherein the oligonucleotide linker sequences are double-stranded and are cleaved with a restriction endonuclease such that a cohesive end is generated prior to annealing with the cloning vector.

14. The method according to claim 1 further comprising the step of subjecting the annealed product to an enzyme capable of forming a covalent 3'"'"'-5'"'"' phosphodiester bond between the single-stranded RNA sequence and the cloning vector to form a recombinant molecule.

15. The method according to claim 1 further comprising the step of converting the RNA sequence to a cDNA/RNA hybrid molecule.

16. A method for cloning an RNA sequence, comprising:

(a) inserting the single-stranded RNA sequence into a cloning vector by annealing both termini of the single-stranded RNA sequence with a linear double-stranded cloning vector having single-stranded termini complementary to both termini of the single-stranded RNA sequence to form an annealed product, in which the complementary termini of the single-stranded RNA sequence were formed by attaching oligonucleotide linkers to the RNA sequence by means of an RNA ligase;

(b) converting the RNA sequence to a cDNA/RNA hybrid to form a recombinant molecule;

(c) introducing the recombinant molecule containing the cDNA/RNA hybrid into a compatible unicellular organism capable of replicating the recombinant molecule to form transformants; and

(d) growing the transformants under appropriate nutrient conditions.

17. A method for the insertion of a single-stranded RNA sequence into a cloning vector, comprising:

annealing the single-stranded RNA sequence with a linear double-stranded cloning vector having a 5'"'"'-single-stranded terminus complementary to the 5'"'"'-terminus of the single-stranded RNA sequence to form an annealed product, in which the 5'"'"'-complementary terminus of the single-stranded RNA sequence was formed by attaching an oligonucleotide linker to the single-stranded RNA sequence by means of an RNA ligase.

18. The method according to claim 17 wherein said RNA ligase is T4 RNA ligase.

19. The method according to claim 18 wherein said oligonucleotide linker attached to the 5'"'"'-terminus of the RNA sequence is composed of a mixture of DNA and RNA such that the 3'"'"'-terminus of the oligonucleotide linker is a ribonucleotide.

20. The method according to claim 18 wherein said oligonucleotide linker attached to the 5'"'"'-terminus of the RNA sequence is composed of RNA.

21. The method according to claim 17 further comprising the step of subjecting the annealed product to an enzyme capable of forming a covalent 3'"'"'-5'"'"'-phosphodiester bond between the single-stranded RNA sequence and the cloning vector.

22. The method according to claim 21 wherein said RNA ligase is T4 RNA ligase and a covalent bond is formed between the 3'"'"'-terminus of the single-stranded RNA sequence and the cloning vector to form a recombinant molecule.

23. The method according to claim 22 further comprising the step of converting the RNA sequence to a cDNA/RNA hybrid molecule.

24. A method for cloning an RNA sequence, comprising:

(a) inserting the single-stranded RNA sequence into a cloning vector by annealing the single-stranded RNA sequence with the linear double-stranded cloning vector having a 5'"'"'-single-stranded terminus complementary to the 5'"'"'-terminus of the single-stranded RNA sequence to form an annealed product;

(b) forming a covalent bond between the 3'"'"'-terminus of the single-stranded RNA sequence and the cloning vector by means of an RNA ligase, to form a recombinant molecule;

(c) converting the RNA sequence of the recombinant molecule to a cDNA/RNA hybrid to form a double-stranded recombinant molecule;

(d) introducing the double-stranded recombinant molecule containing the cDNA/RNA hybrid into a compatible unicellular organism capable of replicating the recombinant molecule to form transformants; and

(e) growing the transformants under appropriate nutrient conditions.

25. A method for the insertion of a single-stranded RNA sequence into a cloning vector, comprising:

(a) annealing the 3'"'"'-terminus of the single-stranded RNA sequence with a linear double-stranded cloning vector having a first 3'"'"'-single-stranded terminus complementary to the 3'"'"'-terminus of the single-stranded RNA sequence and a second 3'"'"'-single-stranded terminus composed of RNA; and

(b) forming a covalent bond between the 5'"'"'-terminus of the single-stranded RNA sequence and the second 3'"'"'-terminus of the cloning vector by means of an RNA ligase.

26. The method according to claim 25 wherein said first 3'"'"'-single-stranded terminus of the cloning vector is formed by ligating an oligonucleotide linker to a linear double-stranded cloning vector.

27. The method according to claim 25 wherein said first 3'"'"'-single-stranded terminus of the cloning vector is formed by attaching nucleotides to a linear double-stranded cloning vector by means of terminal deoxynucleotidyl transferase.

28. The method according to claim 25 wherein said second 3'"'"'-single-stranded terminus of the cloning vector is formed by attaching RNA to a linear double-stranded cloning vector by means of terminal deoxynucleotidyl transferase.

29. The method according to claim 25 wherein said RNA ligase is T4 RNA ligase and a covalent bond is formed between the 5'"'"'-terminus of single-stranded RNA sequence and said second 3'"'"'-terminus of the cloning vector to form a recombinant molecule.

30. The method according to claim 29 further comprising the step of converting the RNA sequence to a cDNA/RNA hybrid molecule.

31. A method for cloning an RNA sequence comprising:

(a) inserting the single-stranded RNA sequence into a cloning vector by annealing the single-stranded RNA sequence with the cloning vector having a first 3'"'"'-single-stranded terminus complementary to the 3'"'"'-terminus of the single-stranded RNA sequence, and a second 3'"'"'-single-stranded terminus composed of RNA to form an annealed product;

(b) forming a covalent bond between the 5'"'"'-terminus of the single-stranded RNA sequence and the second 3'"'"'-terminus of the cloning vector by means of an RNA ligase to form a recombinant molecule;

(c) converting the RNA sequence of the recombinant molecule to a cDNA/RNA hybrid to form a double-stranded recombinant molecule;

(d) introducing the double-stranded recombinant molecule containing the cDNA/RNA hybrid into a compatible unicellular organism capable of replicating the recombinant molecule to form transformants; and

(e) growing the transformants under appropriate nutrient conditions.

32. A method for the insertion of a single-stranded RNA sequence into a cloning vector, comprising:

(a) circularizing the RNA sequence by means of an enzyme capable of ligating RNA;

(b) synthesizing a cDNA strand which is complementary to the circularized RNA sequence to form a CDNA/RNA circular hybrid molecule;

(c) ligating the cDNA strand and removing the RNA portion of the circular hybrid molecule;

(d) syntesizing a second DNA strand which is complementary to the cDNA strand to form a circular double-stranded DNA molecule;

(e) cleaving the circular double-stranded DNA molecule by means of a restriction endonuclease capable of cleaving said circular DNA to produce a linear double-stranded DNA molecule having single-stranded termini; and

(f) annealing the linear double-stranded DNA molecule with a linear double-stranded cloning vector having complementary single-stranded termini to form an annealed product.

33. The method according to claim 32 wherein said enzyme capable of ligating RNA is T4 RNA ligase.

34. The method according to claim 32 further comprising the step of subjecting the annealed product to an enzyme capable of forming covalent 3'"'"'-5'"'"' phosphodiester bonds between the double-stranded DNA molecule and the double-stranded cloning vector to form a recombinant molecule.

35. A method for cloning an RNA sequence, comprising:

(a) circularizing the RNA sequence by means of an enzyme capable of ligating RNA;

(b) synthesizing a cDNA strand which is complementary to the circularized RNA sequence to form a cDNA/RNA circular hybrid molecule;

(c) ligating the cDNA strand and removing the RNA portion of the circular hybrid molecule;

(d) synthesizing a second strand of DNA which is complementary to the cDNA strand to form a circular double-stranded DNA molecule;

(e) cleaving the circular double-stranded DNA molecule by means of a restriction-endonuclease capable of cleaving said circular DNA to produce a linear double-stranded molecule having single-stranded termini;

(f) annealing the linear double-stranded DNA molecule with a linear double-stranded cloning vector having complementary single-stranded termini to form an annealed product;

(g) forming a covalent 3'"'"'-5'"'"'phosphodiester bond between the double-stranded DNA molecule and the double-stranded cloning vector to form a recombinant molecule;

(h) introducing the recombinant molecule into a compatible unicellular organism capable of replicating the recombinant molecule to form transformants; and

(i) growing said transformants under appropriate nutrient conditions.

36. A method for the insertion of a single-stranded RNA sequence into a cloning vector, comprising:

(a) attaching to said RNA sequence an oligonucleotide linker by means of an enzyme capable of ligating RNA to form an RNA-linker product, wherein said linker contains a deoxyribonucleotide sequence encoding a restriction endonuclease recognition site and a 3'"'"'-terminal ribonucleotide;

(b) circularizing the RNA-linker product by means of an enzyme capable of ligating RNA;

(c) synthesizing a cDNA strand which is complementary to the circularized RNA sequence to form a cDNA/RNA circular hybrid molecule containing a duplex DNA site that encodes a restriction enzyme recognition site;

(d) cleaving the circular hybrid molecule at said duplex DNA site by means of a restriction endonuclease capable of cleaving said duplex DNA site to form a linear hybrid molecule having single-stranded termini; and

(e) annealing the linear hybrid molecule with a linear double-stranded cloning vector having complementary termini to form an annealed product.

37. The method according to claim 36 wherein said enzyme is T4 RNA ligase.

38. The method according to claim 36 further comprising the step of subjecting the annealed product to an enzyme capable of forming covalent 3'"'"'-5'"'"'phosphodiester bonds between the hybrid molecule and the double-stranded cloning vector to form a recombinant molecule.

39. A method for cloning an RNA sequence, comprising:

(a) attaching to said RNA sequence an oligonucleotide linker by means of an enzyme capable of ligating RNA to form an RNA-linker product, wherein said linker contains a deoxyribonucleotide sequence encoding a restriction endonuclease recognition site and a 3'"'"'-terminal ribonucleotide;

(b) circularizing the RNA-linker product by means of an enzyme capable of ligating RNA;

(c) synthesizing a cDNA strand which is complementary to the circularized RNA sequence to form a cDNA/RNA circular hybrid molecule containing a duplex DNA site that encodes a restriction enzyme recognition site;

(d) cleaving the circular hybrid molecule at said duplex DNA site by means of a restriction endonuclease capable of cleaving said duplex DNA site to form a linear hybrid molecule having single-stranded termini;

(e) annealing the linear hybrid molecule with a linear double-stranded cloning vector having complementary single-stranded termini to form an annealed product;

(f) forming a covalent 3'"'"'-5'"'"'phosphodiester bond between the hybrid molecule and the double-stranded cloning vector to form a recombinant molecule;

(g) introducing the recombinant molecule into a compatible unicellular organism capable of replicating the recombinant molecule to form transformants; and

(h) growing said transformants under IIIIIIappropriate nutrient conditions.