# US 9,752,172 B2 · Methods and kits for sense RNA synthesis

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- Number: US 9,752,172 B2
- Document ID: US9752172B2
- Status: Granted
- Kind: B2
- Application number: 13/332,955
- Filed: 2011-12-21
- Published: 2012-05-31
- Granted: 2017-09-05
- Earliest claimed: 2005-06-10
- Assignees: Genisphere, LLC
- Applicants: Robert C. Getts; Kelly Sensinger; James Kadushin
- Inventors: Robert C. Getts; Kelly Sensinger; James Kadushin
- Web page: https://patentgrep.com/patent/US9752172B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The kit of claim 1, further comprising a reverse transcriptase.

### Claim 3 (depends on 1)

The kit of claim 1, further comprising a DNA polymerase.

### Claim 4 (depends on 1)

The kit of claim 1, further comprising a terminal deoxynucleotidyl transferase.

### Claim 5 (depends on 1)

The kit of claim 1, further comprising one or more RNA polymerases.

### Claim 6 (depends on 1)

The kit of claim 1, further comprising an RNase inhibitor.

### Claim 7 (depends on 1)

The kit of claim 1, further comprising a polyA polymerase.

### Claim 8 (depends on 1)

The kit of claim 1, further comprising buffers, primers, nucleotides, labeled nucleotides, or RNase-free water.

### Claim 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.

### Claim 10 (depends on 9)

The kit of claim 9, further comprising a tailing buffer.

### Claim 11 (depends on 9)

The kit of claim 9, further comprising at least one dNTP and/or at least one rNTP.

### Claim 12 (depends on 9)

The kit of claim 9, further comprising RNase H.

### Claim 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.

### Claim 14 (depends on 13)

The oligonucleotide of claim 13, wherein the first and second RNA polymerase recognition sequences are recognized by a bacteriophage RNA polymerase.

### Claim 15 (depends on 14)

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.

### Claim 16 (depends on 13)

The oligonucleotide of claim 13 which has a 3′ end blocked with a 3′ amino modifier, a 3′ deoxyterminator or a 3′ dideoxyterminator.

### Claim 17 (depends on 13)

The oligonucleotide of claim 13, wherein the 3′ homopolymeric sequence is complementary to a 3′ oligodeoxynucleotide tail of a cDNA.

### Claim 18 (depends on 17)

The oligonucleotide of claim 17, wherein the 3′ homopolymeric sequence comprises thymidine bases.