Method of identification and cloning differentially expressed messenger RNAs

Abstract

A method of identification of differentially expressed messenger RNA (mRNA) which consists of synthesizing from a set of sequences of mRNA sets of fragments of complementary DNA (cDNA), which are separated with the aid of gel electrophoresis and the pictures of separation of the cDNA from different types of cells are compared and fragments with differential signal intensity are identified. For formation of the set of fragments the cDNA is cleaved with the aid of restriction nucleases. A method of cloning of differentially expressed mRNAs consists of synthesizing from sets of sequences of mRNAs from different types of cells sets of fragments of complementary DNA (cDNA) which are separated with the aid of gel electrophoresis, the pictures of the separation of the cDNA from different types of cells are compared, fragments of cDNA with different signal intensities are separated from the gel, amplified with the aid of a polymerase chain reaction and cloned to a plasmid or phage vector. For the formation of the set of fragments one carried out cleavage of the cDNA with the aid of restriction endonucleases and uses only those fragments of cDNA that correspond to the 3′ or 5′ and regions of the mRNAs.

1 . A method of identification of differentially expressed messenger RNA (mRNA) which consists of synthesizing from sets of mRNA sets of fragments of complementary DNA (cDNA), which are separated with the aid of gel electrophoresis, the separation pictures of the cDNA from different types of cells are compared and one identifies fragments with differential intensity of signals, and which is distinguished by the fact that, for the formation of a set of fragments, cleavage of the cDNA is carried out with the aid of restriction endonucleases.  
     
     
         2 . A method as in  claim 1 , which is distinguished by the fact that for creation of the set of fragments of cDNA one uses only those restriction fragments that correspond to the 3′ or 5′ end regions of the mRNA and the fragments of cDNA are labeled by introducing at one end a radioactive label, a chemical grouping or specific sequence of nucleotides.  
     
     
         3 . A method as in  claim 2 , which is distinguished by the fact that for creation of the set of fragments of cDNA one uses specific immobilization of end fragments on a solid carrier.  
     
     
         4 . A method as in  claim 3 , which is distinguished by the fact that formation of the seat of fragments of cDNA is done by means of ligation, to end fragments of cDNA that are immobilized on a solid carrier and that correspond to the 3′ or 5′ ends of the mRNA, of a double-chain adaptor or single-chain oligonucleotide and reamplification of the set of fragments by means of a polymerase chain reaction.  
     
     
         5 . A method as in  claim 4 , which is distinguished by the fact that for equalization of the degree of representation of different sequences in the set of fragments single or multiple removal of the most represented sequences in the set of fragments of cDNA is done by means of hybridization of nonimmobilized chains of fragments of cDNA with complementary chains immobilized on a solid carrier.  
     
     
         6 - 16 . (canceled)  
     
     
         17 . A method of cloning of differentially expressed mRNAs which consists of synthesizing from a set of sequences of mRNAs from different types of cells sets of fragments of complementary DNA (cDNA) which are separated with the aid of gel electrophoresis, the pictures of separation of the cDNA from different types of cells are compared, the fragments of cDNA with different signal intensities are separated from the gel, amplified with the aid of a polymerase chain reaction and cloned to a plasmid or phage vector, and which is distinguished by the fact that, for the formation of the set of fragments one carries out cleavage of the cDNA with the aid of restriction endonucleases and uses only those fragments of cDNA that correspond to the 3′ or 5′ end regions of the mRNAs.