Methods and kits for sense RNA synthesis
Methods and kits are provided for performing multiple rounds of sense RNA synthesis. The sense RNA molecules can be used in various research and diagnostic applications, such as gene expression studies involving nucleic acid microarrays.
1. A kit for performing multiple rounds of synthesis of at least one sRNA molecule, comprising:
a) as a first oligonucleotide component, a single stranded tandem promoter template comprising a first RNA polymerase recognition sequence, at least a second different RNA polymerase recognition sequence 3′ to said first recognition sequence, and a 3′ homopolymeric sequence about 10 to about 30 nucleotides in length, wherein said single stranded tandem promoter template corresponds to a non-template strand of the first and second RNA polymerase recognition sequences and is blocked at its 3′ end such that the single stranded tandem promoter template is not extendable with DNA polymerase;
b) as a second oligonucleotide component, a single stranded promoter oligonucleotide complementary to said second RNA polymerase recognition sequence; and
c) instructional materials for performing multiple rounds of synthesis of at least one sRNA molecule using said promoter template and promoter oligonucleotide.
2. The kit of claim 1 , further comprising a reverse transcriptase.
3. The kit of claim 1 , further comprising a DNA polymerase.
4. The kit of claim 1 , further comprising a terminal deoxynucleotidyl transferase.
5. The kit of claim 1 , further comprising one or more RNA polymerases.
6. The kit of claim 1 , further comprising an RNase inhibitor.
7. The kit of claim 1 , further comprising a polyA polymerase.
8. The kit of claim 1 , further comprising buffers, primers, nucleotides, labeled nucleotides, or RNase-free water.
9. A kit for performing multiple rounds of synthesis of at least one sRNA molecule comprising the following components and reagents:
a) an oligo dT sequence specific RT primer;
b) a random sequence specific RT primer;
c) a terminal deoxynucleotidyl transferase;
d) as a first oligonucleotide component, a single stranded tandem promoter template comprising a T7 RNA polymerase recognition sequence, a T3 RNA polymerase recognition sequence 3′ to said T7 recognition sequence, and a 3′ homopolymeric sequence about 10 to about 30 nucleotides in length, wherein said single stranded tandem promoter template corresponds to a non-template strand of the T7 and T3 RNA polymerase recognition sequences and is blocked at its 3′ end such that the single stranded tandem promoter template is not extendable with DNA polymerase;
e) Klenow enzyme;
f) T7 RNA polymerase;
g) a second round sequence specific RT primer containing a nucleotide sequence corresponding to a sequence at a 5′ end of the oligo dT and random sequence specific RT primers;
h) as a second oligonucleotide component, a T3 promoter oligonucleotide complementary to the T3 RNA polymerase recognition sequence of the single stranded promoter template;
i) a T3 RNA polymerase; and
instructional materials for producing sRNA molecules using the components and reagents of said kit.
10. The kit of claim 9 , further comprising a tailing buffer.
11. The kit of claim 9 , further comprising at least one dNTP and/or at least one rNTP.
12. The kit of claim 9 , further comprising RNase H.
13. A single stranded oligonucleotide comprising, in tandem, a first RNA polymerase recognition sequence, at least a second different RNA polymerase recognition sequence 3′ to said first recognition sequence, and a 3′ homopolymeric sequence about 10 to about 30 nucleotides in length, wherein said single stranded oligonucleotide corresponds to a non-template strand of the first and second RNA polymerase recognitions sequences and is blocked at its 3′ end such that the single stranded oligonucleotide is not extendable with DNA polymerase.
14. The oligonucleotide of claim 13 , wherein the first and second RNA polymerase recognition sequences are recognized by a bacteriophage RNA polymerase.
15. The oligonucleotide of claim 14 , wherein the first and second RNA polymerase recognition sequences are each independently selected from the group consisting of T7, T3 and SP6 RNA polymerase recognition sequences.
16. The oligonucleotide of claim 13 which has a 3′ end blocked with a 3′ amino modifier, a 3′ deoxyterminator or a 3′ dideoxyterminator.
17. The oligonucleotide of claim 13 , wherein the 3 ′ homopolymeric sequence is complementary to a 3′ oligodeoxynucleotide tail of a cDNA.
18. The oligonucleotide of claim 17 , wherein the 3′ homopolymeric sequence comprises thymidine bases.