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 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.

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′ and 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 and fragments on a solid carrier.  
     
     
         4 . A method as in  claim 3 , which is distinguished by the fact that formation of the set of fragments of cDNA is done by means of ligation, to and 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 . A method as in  claim 4  or  5 , which is distinguished by the fact that, with the goal of improving the sensitivity and resolution of the method, the initial set of fragments of cDNA is divided into nonintersecting subsets of fragments.  
     
     
         7 . A method as in  claim 6 , which is distinguished by the fact that the division of the initial set of fragments of cDNA into subsets is done by means of immobilization of the set of fragments labeled at one end, by the other end on a solid phase and successive cleavage with a set of restriction endonucleases.  
     
     
         8 . A method as in  claim 6 , which is distinguished by the fact that the division of the initial set into subsets is done with the aid of several separate amplification reactions with paired combinations of primers corresponding to and sequences that are common for all of the fragments of cDNA, but that contain an additional base of the 3′ and, immediately after the regions that are common for all the fragments of cDNA.  
     
     
         9 . A method as in any of claims  4 - 8 , which is distinguished by the fact that radioactive  32 P or  33 P is used as the label.  
     
     
         10 . A method as in any of claims  4 - 8 , which is distinguished by the fact that a chemical grouping is used as the label and the separated fragments after transfer to a membrane are detected with the aid of a reagent that specifically binds this group and is conjugated with the enzyme, by means of registration of the enzymatic activity of this enzyme.  
     
     
         11 . A method as in any of claims  4 - 8 , which is distinguished by the fact that the separated fragments are transferred to a membrane and after immobilization visualization of the fragments is done by means of hybridization with a labeled oligonucleotide corresponding to the adaptor sequence.  
     
     
         12 . A method as in any of claims  4 - 8 , which is distinguished by the fact that the separated fragments are transferred from membrane and after immobilization visualization of the subsets of separated fragments is done by means of hybridization of the membrane with a set of labeled oligonucleotides that correspond to the adaptor sequence and that carry at the 3′ and one or several additional bases.  
     
     
         13 . A method as in any of claims  4 - 12 , which is distinguished by the fact that the fragments are separated by two-dimensional gel electrophoresis.  
     
     
         14 . A method as in  claim 13 , which is distinguished by the fact that fragments are separated by length in one direction of two-dimensional electrophoresis and by melting profile using a denaturing gradient in the other direction.  
     
     
         15 . A method as in  claim 13 , which is distinguished by the fact that single-chain fragments are separated by length under denaturing conditions in one direction of two-dimensional electrophoresis and according to secondary structure in nondenaturing conditions in the second direction.  
     
     
         16 . A method as in  claim 13 , which is distinguished by the fact that fragments are separated by length in the first direction of two-dimensional electrophoresis and in the switch to the second direction the fragments of cDNA are specifically cleaved in the gel by a set of restriction endonucleases, after which a second separation by length is carried out.  
     
     
         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. 
 Summary  
 A method of identification of differentially express 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 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.