Reciprocal subtraction differential display

Abstract

This invention provides a method for identifying differentially expressed nucleic acids between two samples, comprising: a) selecting a first and second nucleic acid sample; b) producing libraries for the first and second nucleic acid sample; c) performing reciprocal subtraction between the libraries to produce two subtracted libraries; d) amplifying the two subtracted libraries; and e) comparing the two amplified subtracted libraries to identify differentially expressed nucleic acids. Also, this invention provides the above-described method, wherein the 3′ primer used in the PCR amplification is an oligo dT 3′ primer. This invention also provides the above-described methods, wherein the comparing of step e comprises using a gel to separate the nucleic acids from both of the libraries. This invention provides the isolated nucleic acid identified by the the above-described methods, wherein the nucleic was not previously known to be differentially expressed between the two samples.

What is claimed is:  
     
         1 . A method for identifying differentially expressed nucleic acids between two samples, comprising: 
 a. selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids;    b. performing reciprocal subtraction between the nucleic acid samples to produce two subtracted nucleic acid samples;    c. amplifying the two subtracted nucleic acid samples; and    d. comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.    
     
     
         2 . A method for identifying differentially expressed nucleic acids between two samples, comprising: 
 a. selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids;    b. amplifying the two nucleic acid samples;    c. performing reciprocal subtraction between the amplified nucleic acid samples to produce two subtracted nucleic acid samples; and    d. comparing the two subtracted nucleic acid samples to identify differentially expressed nucleic acids.    
     
     
         3 . The method of  claim 2 , wherein the two subtracted nucleic acid samples from step c are amplified prior to the comparing of step d.  
     
     
         4 . The method of  claim 1  or  2 , wherein the each of the nucleic acid samples comprises a library of nucleic acids.  
     
     
         5 . The method of  claim 1  or  2 , wherein the nucleic acid samples are mRNA or cDNA derived from mRNA.  
     
     
         6 . The method of  claim 1  or  2 , wherein the nucleic acid samples are obtained from total RNA from E11 and E11-NMT cells.  
     
     
         7 . The method of  claim 1  or  2 , wherein the first and second nucleic acid samples are obtained from cells that differ in their exposure to external factors or in their gene expression.  
     
     
         8 . The method of  claim 1  or  2 , wherein the first and second nucleic acid samples are obtained from cells in different developmental stages.  
     
     
         9 . The method of  claim 1  or  2 , wherein the amplifying of step (d) comprises PCR amplification.  
     
     
         10 . The method of  claim 9 , wherein the PCR amplification uses a set of random primers.  
     
     
         11 . The method of  claim 9 , wherein the 3′ primer used in the PCR amplification is a single anchor oligo dT 3′ primer.  
     
     
         12 . The method of  claim 9 , wherein the 5′ primer is an arbitrary primer.  
     
     
         13 . The method of  claim 1  or  2 , wherein the comparing of step (e) comprises using a gel to separate the nucleic acids from both of the libraries.  
     
     
         14 . The method of  claim 1  or  2 , further comprising PCR amplifying the first and second nucleic acid samples.  
     
     
         15 . The method of  claim 1  or  2 , further comprising reamplifying differentially expressed nucleic acids.  
     
     
         16 . The method of  claim 1  or  2 , wherein the comparing of step (e) comprises comparing the quantities of the two amplified differentially expressed nucleic acids.  
     
     
         17 . The method of  claim 1  or  2 , wherein differences in the quantities of nucleic acid between the two subtracted libraries are electronically quantified.  
     
     
         18 . The method of  claim 1  or  2 , wherein the libraries of step (b) are constructed with λ-ZAP cDNA library kits.  
     
     
         19 . The isolated nucleic acid identified by the method of  claim 1  or  2 , wherein the nucleic acid was not previously known.  
     
     
         20 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 12.  
     
     
         21 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 13.  
     
     
         22 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 23.  
     
     
         23 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 24.  
     
     
         24 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 25.  
     
     
         25 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 26.  
     
     
         26 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 27.  
     
     
         27 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 28.  
     
     
         28 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PSGen 29.  
     
     
         29 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 13.  
     
     
         30 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 14.  
     
     
         31 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 15.  
     
     
         32 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 24.  
     
     
         33 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 28.  
     
     
         34 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 32.  
     
     
         35 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 42.  
     
     
         36 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 43.  
     
     
         37 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 44.  
     
     
         38 . The isolated nucleic acid of  claim 19 , wherein the isolated nucleic acid is the nucleic acid designated PEGen 48.  
     
     
         39 . The isolated nucleic acid molecule of  claim 19  which comprises: 
 (a) one of the nucleic acid sequences as set forth in FIG. 35;  
 (b) a sequence being degenerated to a sequence of (a) as a result of the genetic code;  
 (c) a sequence encoding one of the amino acid sequences as set forth in FIG. 35.  
 (d) a sequence of at least 12 nucleotides capable of specifically hybridizing to the sequence of (a), (b) or (c)  
 
     
     
         40 . A purified polypeptide comprising one of the amino acid sequence as set forth in FIG. 35.