Methods for generating double stranded DNA comprising a 3' single stranded portion and uses of these complexes for recombination
First Claim
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1. A method for generating a hybrid nucleic acid, said method comprising:
- (a) preparing a complex of first and second primer extension products said complex comprising a 3′
single stranded portion, wherein said complex comprising a 3′
single stranded portion is prepared according to a method comprising;
(i) extending a first primer hybridized to a target RNA with an enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;
(ii) cleaving RNA in the complex of step (i);
(iii) extending a second primer hybridized to the first primer extension product with an enzyme comprising DNA-dependent DNA polymerase activity and an enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(iv) cleaving RNA from the composite primer in the complex of step (iii) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that a complex comprising hybridized first and second primer extension product comprising a 3′
single stranded portion is generated; and
(b) hybridizing the 3′
single stranded portion of the complex of first and second primer extension products with a recipient nucleic acid molecule;
whereby a hybrid nucleic acid is generated.
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Abstract
Methods of recombining nucleic acids are provided. In particular, methods for the production of partially double stranded nucleic acids comprising a 3′ overhang from an RNA target and use in methods of recombining polynucleotides is described. These methods do not require thermocycling. The present invention also provides methods of recombining and selection which allow for identification of proteins comprising improved or desired characteristics.
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29 Claims
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1. A method for generating a hybrid nucleic acid, said method comprising:
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(a) preparing a complex of first and second primer extension products said complex comprising a 3′
single stranded portion, wherein said complex comprising a 3′
single stranded portion is prepared according to a method comprising;
(i) extending a first primer hybridized to a target RNA with an enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;
(ii) cleaving RNA in the complex of step (i);
(iii) extending a second primer hybridized to the first primer extension product with an enzyme comprising DNA-dependent DNA polymerase activity and an enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(iv) cleaving RNA from the composite primer in the complex of step (iii) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that a complex comprising hybridized first and second primer extension product comprising a 3′
single stranded portion is generated; and
(b) hybridizing the 3′
single stranded portion of the complex of first and second primer extension products with a recipient nucleic acid molecule;
whereby a hybrid nucleic acid is generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method of making a hybrid nucleic acid, said methods comprising:
- hybridizing a 3′
single stranded portion of a complex of first and second primer extension products with a recipient nucleic acid molecule, whereby a hybrid nucleic acid is generated, wherein said complex of first and second primer extension products is prepared according to a method comprising;
(i) extending a first primer hybridized to a target RNA with an enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;
(ii) cleaving RNA in the complex of step (i);
(iii) extending a second primer hybridized to the first primer extension product with an enzyme comprising DNA-dependent DNA polymerase activity and an enzyme comprising RNA-dependent polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(iv) cleaving RNA from the composite primer in the complex of step (iii) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that a complex comprising hybridized first and second primer extension product comprising a 3′
single stranded portion is generated.
- hybridizing a 3′
Specification