Ribocloning: Recombinant DNA Construction using Primers with RIBO Bases

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.

1 . 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 themostable DNA polymerase under PCR conditions which comprise annealing/extension steps of 15 to 30 minutes. 
     
     
         2 . A method of  claim 1  wherein at one or each end of a PCR amplimer, only two to four 40-60 mers, such as 42 mers, overlap each other and a target DNA to be amplified and a PCR primer at that end or each end of the amplimer. 
     
     
         3 . A method of  claim 2  wherein at one or each end of a PCR amplimer, only one 40-60 mer, such as a 50 mer, overlaps both a target DNA to be amplified and a PCR primer at that end or at each of the amplimer.