Methods for making nucleic acids

• US 6,114,152 A
• Filed: 03/27/1998
• Issued: 09/05/2000
• Est. Priority Date: 12/12/1997
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Serafini, Tito, Lin, David, Ngai, John, Luu, Percy
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): SCHMIDT, MARY M
• Application Number:
  US09/049,806
• Time in Patent Office: 893 Days
• Field of Search: 435/91.2, 435/91.3, 435/91.1, 435/91.5, 435/91.51, 435/91.52, 536/23.1, 536/24.3, 536/24.1
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

Nucleic acids are made by adding a known nucleotide sequence to the 3'"'"' end of a first RNA having a known sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. In one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5'"'"' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3'"'"' end, the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence, the cDNA is converted to double-stranded cDNA by a polymerase initiating from a noncovalently joined duplex region, and the double-stranded cDNA is transcribed to form one or more third RNAs.

Claims

1. A method for making a nucleic acid comprising the steps of adding a known nucleotide sequence to the 3'"'"' end of a first RNA having a known sequence at the 5'"'"' end to form a second RNA and reverse transcribing the second RNA to form a cDNA.

2. A method according to claim 1, wherein the adding step comprises contacting the first RNA with at least one of (a) a nucleotide and polyadenyltransferase and (b) an oligonucleotide and a ligase, whereby said polyadenyltransferase and/or said ligase adds said known nucleotide to the 3'"'"' end of said first RNA to form said second RNA.

3. A method according to claim 1, wherein the known sequence at the 3'"'"' end comprises, a poly(A) sequence.

4. A method according to claim 1, wherein the known sequence at the 5'"'"' end comprises at least one of (a) a poly(T) or poly(A) sequence and (b) an internal sequence of an mRNA or the complement thereof.

5. A method according to claim 1, wherein the known sequence at the 5'"'"' end comprise a poly(T) sequence and an RNA polymerase promoter sequence.

6. A method according to claim 1, wherein the known sequence at the 3'"'"' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence.

7. A method according to claim 1, wherein the known sequence at the 3'"'"' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence, wherein the poly(T) sequence is covalently joined to at least one of a RNA polymerase promoter sequence and a primer sequence.

8. A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA.

9. A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA by a method comprising the step of contacting the RNA with at least one of an RNase H, a denaturant, and an alkali.

10. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA.

11. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA and the conversion is initiated at a noncovalently joined duplex region.

12. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with as RNase H and contacting the single-stranded cDNA with a DNA polymerase whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region.

13. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

14. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA and an RNA polymerase promoter, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

15. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA and an RNA polymerase promoter comprising a natural class III T7 RNA polymerase promoter sequence, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

16. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an at oligonucleotide primer comprising a sequence complementary to the 3'"'"' end of the single-stranded cDNA and an RNA polymerase promoter comprising SEQ ID NO:

1 joined to an upstream flanking sequence of about 3 to 100 nucleotides, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3'"'"' end of the single-stranded cDNA and the oligonucleotide primer.

17. A method according to claim 1, further comprising the step of repeatedly transcribing the cDNA to form a plurality of third RNAs.

18. A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA, and the method further comprises the step of repeatedly transcribing the double-stranded cDNA to form a plurality of third RNAs.

19. A method according to claim 1, wherein the first RNA is made by amplifying a mRNA.

20. A method according to claim 1, wherein the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.

21. A method according to claim 1, wherein the adding step comprises contacting the first RNA with a nucleotide and polyadenyltransferase.