Ribocloning: recombinant DNA construction using primers with ribo bases
AssigneeDna Polymerase Technology, Inc.
InventorWayne Morris Barnes
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Abstract
The present invention provides methods of linking nucleic acids without the use of restriction enzymes or any joining enzyme such as ligase. More specifically, the present invention provides methods for cloning or rearranging a double-stranded target DNA which is a PCR product into a double-stranded vector DNA which is a PCR product, said DNAs being amplified using primers that contain at least one ribonucleotide, preferably at or near the respective 3′ ends of the primers, such that RNA-specific cleavage, preferably by RNAse A, will allow release of the primers to create long matching 3′-sticky ends.
Claims25
A method of cloning a double-stranded target DNA into a double stranded vector DNA, said method comprising:
- a) generating, for instance by PCR-amplification, double-stranded target DNA molecules by extension of target-specifying (i.e. pair of) primers that each contain at least one ribonucleotide, at one or more positions from 5 nucleotides from their 5′ ends to at their 3′-ends, wherein one of said target-specifying primers is complementary to one of vector-specifying (i.e. pair of) primers that would direct PCR-amplification of vector, and the other of said target primers is complementary to the other of said vector-specifying primers;
- b) exposing the target DNA to RNA-specific cleaving conditions that result in cleavage at the one or more ribonucleotides contributed by each primer, thereby readily allowing the generation of double-stranded DNA with 3′-overhangs by melting off the primer portion which is 5′ to the most 3′-ribonucleotide that was cleaved;
- c) similarly providing a double-stranded vector DNA with 3′-overhangs that are specifically complementary to the 3′-overhangs of the cleaved target DNA;
- and
- d) incubating said cleaved target DNA and said vector DNA together under conditions that result in annealing of the complementary ends thereof to form the desired recombinant.
The method of claim 14, wherein the RNAse is RNAse T1.
The use of an archebacterial DNA polymerase such as Vent, Deep Vent, Pfu, or Pwo, together with a minority component at a relative amount such as {fraction (1/10)} to {fraction (1/500)} of Taq or Klentaq DNA polymerase, to catalyze PCR-amplification.
A method of gene assembly, wherein disconnected, but adjacent on the designed sequence, 40-60 mers of DNA that overlap with staggered complementary oligomers of the same size to form a gene, are provided at a concentration of 1 to 10 nanomolar, and cycled with a thermostable DNA polymerase under PCR conditions which comprise annealing/extension steps of 15 to 30 minutes.
Dependencies are inferred from the claim text.