Methods for amplification of nucleic acid sequences using staggered ligation
Methods and kits are provided for producing sense RNA molecules. The sense RNA molecules are prepared by: providing a single stranded cDNA molecule having 5′ and 3′ ends; attaching an oligodeoxynucleotide tail to the 3′ end of said single stranded cDNA molecule; providing a double stranded RNA polymerase promoter having a sense strand and antisense strand, wherein the sense strand comprises a single stranded 3′ overhang comprising a sequence complementary to said oligodeoxynucleotide tail; annealing said double stranded RNA polymerase promoter to said oligodeoxynucleotide tail by complementary base pairing with said 3′ overhang sequence; ligating the 5′ end of the antisense strand of said double stranded RNA polymerase promoter to the 3′ end of said oligodeoxynucleotide tail; and initiating RNA transcription using an RNA polymerase which recognizes said double stranded promoter, thus producing a sense RNA molecule (sRNA).
1. A method for producing a sense RNA molecule, comprising:
providing a single stranded cDNA molecule having 5′ and 3′ ends;
attaching an oligodeoxynucleotide tail to the 3′ end of said single stranded cDNA molecule;
providing a double stranded RNA polymerase promoter having a sense strand and antisense strand, wherein the sense strand comprises a single stranded 3′ overhang comprising a sequence complementary to said oligodeoxynucleotide tail;
annealing said double stranded RNA polymerase promoter to said oligodeoxynucleotide tail by complementary base pairing with said 3′ overhang sequence;
ligating the 5′ end of the antisense strand of said double stranded RNA polymerase promoter to the 3′ end of said oligodeoxynucleotide tail; and
initiating RNA transcription using an RNA polymerase which recognizes said double stranded promoter, thus producing a sense RNA molecule (sRNA).
2. The method of claim 1 , wherein said attaching comprises providing a mRNA transcript having 5′ and 3′ ends; and synthesizing a single stranded cDNA molecule from said mRNA transcript.
3. The method of claim 2 , wherein synthesis of the single stranded cDNA molecule comprises reacting the mRNA transcript with a RNase H- reverse trancriptase.
4. The method of claim 2 , wherein synthesis of the single stranded cDNA molecule comprises reacting the mRNA transcript with an oligodT primer.
5. The method of claim 2 , wherein synthesis of the single stranded cDNA molecule comprises reacting the mRNA transcript with a random primer.
6. The method of claim 2 , further comprising purifying the single stranded cDNA molecule prior to attaching the oligodeoxynucleotide tail.
7. The method of claim 6 , further comprising degrading the mRNA transcript prior to purifying the single stranded cDNA molecule.
8. The method of claim 6 , wherein the mRNA transcript is not degraded prior to purifying the single stranded cDNA molecule.
9. The method of claim 1 , wherein the oligodeoxynucleotide tail is a homopolymeric tail.
10. The method of claim 9 , wherein the homopolymeric tail is a polydT tail.
11. The method of claim 1 , wherein the oligodeoxynucleotide tail is attached to the 3′ end of the single stranded cDNA molecule using terminal deoxynucleotidyl transferase.
12. The method of claim 1 or 2 , wherein the double stranded RNA polymerase promoter is a T7, T3, or SP6promoter.
13. The method of claim 12 , wherein the double stranded RNA polymerase promoter is a T7 promoter.
14. The method of claim 1 , wherein the single stranded 3′ overhang comprises a sequence of adenosine bases.
15. The method of claim 1 , wherein ligation is performed using T4 DNA ligase.
16. The method of claim 1 , wherein RNA transcription is initiated using T7 RNA polymerase.
17. The method of claim 1 , further comprising synthesizing second strand cDNA prior to initiating RNA transcription.
18. The method of claim 17 , wherein the second strand cDNA is synthesized using DNA polymerase.
19. The method of claim 17 , wherein the second strand cDNA is synthesized by extension of the 3′ overhang of the sense strand of the RNA polymerase promoter.
20. The method of claim 17 , wherein the second strand cDNA is synthesized using a random primer, thus producing random-primed second strand cDNA fragments.
21. The method of claim 20 , wherein the random-primed second strand cDNA fragments are ligated together prior to initiating RNA transcription.
22. The method of claim 1 , further comprising amplifying the resulting sRNA molecule.
23. The method of claim 22 , wherein the sRNA amplification is initiated using a combination of oligodT and random primers.
24. The method of claim 1 , wherein the resulting sRNA molecule comprises a polyA tail.
25. The method of claim 24 , wherein the polyA tail is attached using polyA polymerase.
26. The method of claim 1 , further comprising reverse transcribing the resulting sRNA molecule, thereby producing a single stranded cDNA molecule.
27. The method of claim 26 , wherein the reverse transcription comprises incorporating detectably labeled nucleotides into the single stranded cDNA molecule.
28. The method of claim 27 , wherein the detectably labeled nucleotides comprise a fluorescent dye.
29. The method of claim 28 , wherein the fluorescent dye is cy3 or cy5.
30. The method of claim 26 , further comprising attaching at least one detectable label to the resulting cDNA molecule.
31. A method for probing a nucleic acid microarray, comprising contacting a nucleic acid microarray with detectably labeled cDNA, wherein said detectably labeled cDNA is prepared by the following steps:
providing a single stranded cDNA molecule having 5′ and 3′ ends;
attaching an oligodeoxynucleotide tail to the 3′ end of said single stranded cDNA molecule;
providing a double stranded RNA polymerase promoter having a sense strand and antisense strand, wherein the sense strand comprises a single stranded 3′ overhang comprising a sequence complementary to said oligodeoxynucleotide tail;
annealing said double stranded RNA polymerase promoter to said oligodeoxynucleotide tail by complementary base pairing with said 3′ overhang sequence;
ligating the 5′ end of the antisense strand of said double stranded RNA polymerase promoter to the 3′ end of said oligodeoxynucleotide tail;
initiating RNA transcription using an RNA polymerase which recognizes said double stranded promoter, thus producing a sense RNA molecule (sRNA); and
reverse transcribing a resulting sRNA molecule, thereby producing a single stranded cDNA molecule, wherein the reverse transcribing comprises incorporating detectably labeled nucleotides into the single stranded cDNA molecule.
32. The method of claim 2 , wherein the mRNA transcript is of mammalian origin.
33. The method of claim 2 , wherein the mRNA transcript is of human origin.
34. The method of claim 2 , wherein the mRNA transcript is isolated from a biological source comprising degraded RNA.
35. A kit for producing at least one sRNA molecule, comprising: a double stranded RNA polymerase promoter having a sense strand and antisense strand, wherein the sense strand of said double stranded RNA polymerase promoter comprises a single stranded 3′ overhang sequence; instructional materials for generating sRNA molecules using said double stranded promoter; at least one enzyme for attaching an oligodeoxynucleotide tail onto the 3′ end of a single stranded cDNA molecule, wherein the oligodeoxynucleotide tail is complementary to the single stranded 3′ overhang sequence of said double stranded RNA polymerase promoter; and at least one enzyme for ligating said double stranded promoter onto the 3′ end of said cDNA molecule.
36. The kit of claim 35 , wherein said enzyme for attaching is terminal deoxynucleotidyl transferase and wherein said enzyme for ligating is T4 DNA ligase.
37. The kit of claim 36 , further comprising an oligodT primer; a random primer; dNTPs; and a RNase inhibitor.
38. The kit of claim 37 , further comprising a DNA polymerase.