Restriction display (RD-PCR) of differentially expressed mRNAs
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorsJohn N. Weinstein, John Buolamwini
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Abstract
A method for detecting gene expression in cells by reverse transcribing mRNA molecules into cDNA, cutting the cDNA with at least one restriction endonuclease, adding adaptor sequences to the cDNA fragments and selectively amplifying a subset of the cDNA by a polymerase chain reaction (PCR) to present a two-dimensional display of the DNA fragments or for cloning the DNA fragments into a vector is disclosed. In one embodiment, cDNA corresponding to the 3′ end of the mRNA is amplified and displayed or cloned, whereas in another embodiment, cDNA corresponding to the entire mRNA molecule is amplified and displayed or cloned.
Claims13
A method for characterizing mRNA molecules in a population of mRNAs including the steps of providing purified and isolated mRNA molecules, reverse transcribing a double stranded cDNA from said isolated mRNA molecules, digesting said double stranded cDNA with a restriction endonuclease to produce cDNA fragments in which either one end or both ends of said cDNA fragments have a sequence that specifically hybridizes to adaptor DNA sequences, wherein the improvement comprises:
- after said digesting step, hybridizing adaptor DNA sequences to said cDNA fragments in which both ends of said cDNA fragments have a sequence that specifically hybridizes to said adaptor DNA sequences;
- ligating said adaptor DNA sequences to said cDNA fragments having hybridized adaptor DNA sequences;
- amplifying said cDNA fragments having ligated adaptor DNA sequences by a polymerase chain reaction (PCR) with primer DNA sequences that hybridize to the ends of said cDNA fragments having ligated adaptor DNA sequences, wherein said primer DNA sequences have at least one nucleotide at the 3′ end that specifically hybridizes to a subset of cDNA molecules produced during said reverse transcribing step, thereby producing amplified cDNA fragments;
- and
- detecting the presence of the amplified cDNA fragments, wherein each amplified cDNA fragment corresponds to a mRNA molecule within said population of mRNAs, thereby characterizing mRNA molecules in a population of mRNAs.
A method for characterizing mRNA molecules in a population of mRNAs including the steps of providing purified and isolated mRNA molecules, reverse transcribing a double stranded cDNA from said isolated mRNA molecules, digesting said double stranded cDNA with a restriction endonuclease to produce cDNA fragments in which at least one end of said cDNA fragments has a sequence that specifically hybridizes to an adaptor DNA sequence, wherein the improvement comprises:
- after said digesting step, hybridizing adaptor DNA sequences to said cDNA fragments in which at least one end of said cDNA fragments has a sequence that specifically hybridizes to said adaptor DNA sequences;
- ligating said adaptor DNA sequences to said cDNA fragments having hybridized adaptor DNA sequences;
- amplifying said cDNA fragments having ligated adaptor DNA sequences by a polymerase chain reaction (PCR) with primer DNA sequences that hybridize to the ends of said cDNA fragments having ligated adaptor DNA sequences, wherein said primer DNA sequences have at least one nucleotide at the 3′ end that specifically hybridizes to a subset of cDNA molecules produced during said reverse transcribing step, thereby producing amplified cDNA fragments;
- and
- detecting the presence of the amplified cDNA fragments, wherein each amplified cDNA fragment corresponds to a mRNA molecule within said population of mRNAs, thereby characterizing mRNA molecules in a population of mRNAs,
- and wherein the improvement further comprises, in said reverse transcribing step, priming said isolated mRNA molecules to produce a first strand of cDNA with a primer DNA sequence which comprises, in a 5′ to 3′ orientation,
- a 5′-adaptor DNA sequence;
- one or more nucleotides adjacent to said 5′-adaptor DNA sequence;
- an oligo-dT sequence;
- and
- one or more nucleotides adjacent to said oligo-dT sequence, wherein the nucleotide directly adjacent to said oligo-dT sequence can be C, A or G but not T, and nucleotides 3′ of the nucleotide directly adjacent to said oligo-dT sequence can be C, A, G or T, and wherein said nucleotides adjacent to said oligo-dT sequence specifically hybridize to ribonucleotides occurring 5′ of a poly-(A) sequence In mRNA molecules;
- and
- synthesizing a second strand of cDNA complementary to said first strand of cDNA.
Dependencies are inferred from the claim text.