Production of a random primed DNA for targeted cloning, especially for qualitative gene libraries to analyze central mutations of organisms

摘要

A method to produce a suitable random primed DNA for targeted cloning comprises annealing an RNA to a single stranded oligonucleotide mixture where the 3' end has a random sequence of at least 4 nucleotides and the 5' end has a restriction endonuclease recognition site and performing polymerase chain reaction (PCR) to synthesize first strand cDNA, is new. A method to produce a suitable random primed DNA for targeted cloning, that is characterized by the following steps: (a) annealing an RNA to a single stranded oligonucleotide mixture or to an (m)RNA mixture out of a sample, where the oligonucleotide mixture is characterized in that the 3'-end of the random sequence has a length of at least 4 nucleotides, and that the 5'-end, when it is expressed in a double stranded form, has a recognition site for a restriction endonuclease; (b) production of a first cDNA strand from the oligonucleotide mixture using PCR to synthesize the first cDNA; (c) performing another polymerization reaction to synthesize a second strand of the cDNA; (d) ligation of adaptor-DNA molecules at both ends of the cDNA from step (c); and (e) cleavage of the cDNA molecule from step (d) with a restriction endonuclease which recognizes the recognition site in the 5'-end of the oligonucleotide mixture as above; where the cDNA contains, an end which is defined by the sequence of the adaptor-DNA molecule, the other end has a recognition site for the restriction endonuclease, determined by the oligonucleotide mixture, and where at least one end is an overhanging end. An Independent claim is also included for a kit for performing the method above containing an oligonucleotide mixture as defined above.