Compositions and methods for ordered and continuous complementary DNA (cDNA) synthesis across non-continuous templates
AssigneeThe Regents of the University of California
InventorsKathleen Collins, Heather E. Upton
Life of this patent
Estimating the term…
Abstract
The invention provides compositions and methods for nucleic acid synthesis, including ordered and continuous complementary DNA (cDNA) synthesis across non-continuous templates using a modified eukaryotic non-long terminal repeat reverse transcriptase (non-LTR RT) protein.
Claims16
1-95. (canceled)
An isolated eukaryotic non-long terminal repeat reverse transcriptase (non-LTR RT) protein comprising a truncated N-terminal region, an RNA binding domain, an RT domain, and an endonuclease domain, wherein the endonuclease domain comprises a mutation that abolishes endonuclease function.
A method of extending the 3′ end of a single-stranded or partially single-stranded nucleic acid by at least one nucleotide, the method comprising contacting the single-stranded or partially single-stranded nucleic acid with a non-retroviral RT protein having nucleotide polymerase activity in a buffer comprising manganese ions.
A method of extending the 3′ ends of an A-form nucleic acid duplex by at least one nucleotide, the method comprising contacting the A-form nucleic acid duplex with a non-retroviral RT protein of claim 1 having nucleotide polymerase activity in a buffer comprising manganese ions.
A method of preparing a complementary DNA (cDNA) molecule comprising:
- providing a primer duplex comprising a primer strand and a non-extended strand, wherein the 3′ end of the primer strand comprises a +1 pyrimidine nucleotide overhang;
- providing an RNA template comprising a purine nucleotide at its 3′ end;
- and
- contacting the primer duplex and the RNA template with a RT in a buffer comprising magnesium ions and one or more dNTPs or analogs thereof, wherein the contacting is carried out under conditions effective for production of a cDNA molecule that is substantially complementary to the RNA template.
Dependencies are inferred from the claim text.