Analysis of gene expression by display of 3'-end fragments of cDNAs

Abstract

The present invention is directed to an approach to identify changes in gene expression by selective amplification of 3′ fragments of double stranded cDNAs.

We claim:  
     
         1 . A method for selectively amplifying in a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a 5′ sequence incapable of hybridizing to a polyA tail of thie mRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein n is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other; and  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA.  
 
     
     
         2 . The method of  claim 1 , wherein each oligonucleotide primer in step (a) has a different 5′ sequence.  
     
     
         3 . The method of  claim 1 , wherein the contacting step is performed with a mixture of oligonucleotide primers.  
     
     
         4 . The method of  claim 1 , wherein the oligonucleotide primer of step (a) has one non-poly A nucleotide and the first primer of step (f) has two non-polyA nucleotides.  
     
     
         5 . The method of  claim 4 , wherein the oligonucleotide primer is a mixture of 3 primers having 5′-A-3′, 5′-C-3′, 5′-G-3′ as the non-polyA hybridizing nucleotide and the first primer of step (f) is a mixture of 12 primers having 5′-AA-3′, 5′-AT-3′, 5′-AC-3′, 5′-AG-3′, 5′-CA-3′, 5′-CT-3′, 5′-CC-3′, 5′-CG-3′, 5′-GA-3′, 5′-GT-3′, 5′-GC-3′, and 5′-GG-3′ as the non-polyA hybridizing nucleotides.  
     
     
         6 . The method of  claim 1 , wherein each set of primers in step (f) are used in a separate amplification.  
     
     
         7 . The method of  claim 1 , wherein the 5′ sequence of one or both of the primer sequences in step (f) comprises a recognition sequence for a restriction enzyme.  
     
     
         8 . The method of  claim 1 , wherein the adapter comprises a first portion, wherein the two strands are noncomplementary to each other and a second portion, wherein the two strands are complementary to each other, resulting in a partially hybridized adapter that is Y-shaped.  
     
     
         9 . The method of  claim 8 , wherein one of the two strands of the noncomplementary portion comprises a recognition sequence for a restriction enzyme.  
     
     
         10 . The method of  claim 1 , wherein the mRNAs are isolated from cells or tissue.  
     
     
         11 . The method of  claim 1 , wherein at least one of the primers in step (f) is labeled.  
     
     
         12 . The method of  claim 1   1 , wherein the label is a fluorescent label.  
     
     
         13 . The method of  claim 1 , wherein the cleaving agent is a restriction enzyme.  
     
     
         14 . A method for selectively isolating in a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and a non-polyA nucleotides imediately upstream of the polyA tail, wherein n is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other;  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA; and  
 (g) isolating the amplified fragment.  
 
     
     
         15 . A method for selectively cloning from a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a 5′sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein a is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other;  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least n+l non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA; and  
 (g) cloning the isolated fragment.  
 
     
     
         16 . The method of  claim 15 , wherein the cloning step comprises: 
 (g)(1) digesting the amplified fragments in step (f) with a restriction enzyme, and    (g)(2) ligating the digested fragments to a vector.    
     
     
         17 . The method of  claim 15 , further comprising: 
 (h) determining the DNA sequence of cloned fragments.    
     
     
         18 . A method for analyzing in a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein n is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other;  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the a4apter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA; and  
 (g) isolating the amplified fragment; and  
 (h) analyzing the isolated fragment.  
 
     
     
         19 . The method of  claim 18 , wherein the analyzing step comprises: 
 (h)(1) determining the DNA sequences of the fragments.    
     
     
         20 . The method of  claim 18 , wherein the analyzing step comprises: 
 (h)(1) hybridizing the fragments to nucleic acid molecules.    
     
     
         21 . A method for selectively detecting in a nucleic acid sample DNA fragments having sequence complementary to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein n is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other;  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA; and  
 (g) detecting the amplified fragments.  
 
     
     
         22 . The method of  claim 21 , wherein the detecting step comprises: 
 hybridizing the fragments to nucleic acid molecules.    
     
     
         23 . The method of  claim 22 , wherein the nucleic acid molecules are attached to a silicon wafer or porous glass wafer.  
     
     
         24 . The method of  claim 22 , wherein the nucleic acid molecules are oligonucleotides from about 25 to about 40 nucleotides long.  
     
     
         25 . The method of  claim 22 , wherein the nucleic acid molecules comprise a set of cDNA sequences.  
     
     
         26 . The method of  claim 20 , wherein the fragments are labeled.  
     
     
         27 . A method for comparing the levels of mRNA expression in two cell populations, comprising: 
 selectively amplifying in a nucleic acid sample from each cell population DNA fragments having sequences corresponding to 3′ portions of mRNAs, comprising the steps of.    (a) contacting the mRNAs with oligonucleotide primers comprising a 5′ sequence incapable of hybridizing to a polya tail of the miRNAs, and a 3′ sequence that hybridizes to a portion of the polyA tail of the mRNAs and il non-polya nucleotides immediately upstream of the polyA tail, wherein n is at least one;    (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;    (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;    (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;    (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other;    (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the 5′ sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3′ sequence that hybridizes to a portion of the polyA tail of the mnRNAs and at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA; and    (g) comparing the amounts of amplified fragments obtained in step (f).    
     
     
         28 . The method of  claim 27 , wherein one of the cell populations is treated.  
     
     
         29 . The method of  claim 27 , wherein one of the cell populations is a tumor cell population.  
     
     
         30 . A method for selectively amplifying in a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps of: 
 (a) contacting the mRNAs with oligonucleotide primers comprising a sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein n is at least one;  
 (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer;  
 (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;  
 (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments;  
 (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other; and  
 (f) amplifying the ligated cleaved fragments using a set of primers, in which for each set the first primer comprises the sequence that hybridizes to a portion of the polyA tail of the mRNAs and n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3′ region of an mRNA.