Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-11-12
2002-05-28
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S091200, C435S091520, C536S022100, C536S024200, C536S024300, C536S025300
Reexamination Certificate
active
06395887
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention generally provides methods for determining a signature profile of mRNAs expressed in a cell.
BACKGROUND OF THE INVENTION
Each cell from a larger eukaryote expresses approximately 15,000 genes. Of these. as few as one gene may account for a particular phenotype. The identification of those genes associated with development, differentiation, disease states, and response to cellular environment is crucial for understanding of these phenomena. A powerful approach to analyze genes responsible for various cell states is to identify genes that are expressed at higher or lower levels in one cell as compared to a reference cell. Specifically, effective and efficient methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered cell environments.
Early methods developed to identify and clone such genes were primarily based on the principle of differential or subtractive hybridization (see St. John and Davis,
Cell
16:443, 1979; Sargent and Dawid,
Science
222:135, 1983). Despite the usefulness of these methods, several limitations restrict their widespread utility: only a fraction of the overall changes in gene expression are measured, large amounts of ribonucleic acid (RNA) are necessary, and the procedures are complex and labor intensive.
Recent development of an amplification technique, called differential display, allows a more comprehensive analysis of messenger RNAs (mRNAs) (Liang and Pardee,
Science
257:967, 1992; U.S. Pat. No. 5,262,311). The general strategy is to amplify partial cDNA sequences derived from subsets of mRNAs by reverse transcription and polymerase chain reaction. These partial sequences are generated by using a primer that anneals to the 3′ end of all mRNAs and a short, random sequence primer that anneals to a subset (approximately 50-100) of mRNAs. The amplified products are then separated by gel electrophoresis and visualized. Although this method yields patterns of mRNAs, documented artifacts render interpretation of the results difficult. Such artifacts stem from the use of random sequence primers, which must be annealed at non-stringent conditions. In addition, the cell source of the mRNAs may be relatively scarce. For example, stem cell populations constitute a very small fraction of a tissue. The amount of RNA recovered from a small number of cells may be too low to allow multiple reactions, which are necessary for generating a profile. In such cases, differential display methods, including that described in '311 patent and in Prashar and Weissman,
Proc. Natl. Acad. Sci. USA
93:659, 1996, are difficult to perform.
Thus, there is a need in the art for methods of differential display that bypass such artifactual pitfalls and can be readily performed with small amounts of RNA. The present invention provides such an improved method of differential display as well as other related advantages.
SUMMARY OF THE INVENTION
The present invention generally provides methods for selectively amplifying DNA fragments from nucleic acid samples with sequences corresponding to 3′ ends of mRNAs.
In one aspect, the invention provides such a method 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; 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.
In a preferred embodiment, each oligonucleotide primer in step (a) has a different 5′ sequence. In a related embodiment, the oligonucleotide primer of step (a) has one non-poly A nucleotide (e.g., 5′-A-3′, 5′-C-3′, 5′-G-3′) and the first primer of step (f) has two non-polyA nucleotides (e.g., 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′). The contacting step of the method may also be performed with a mixture of oligonucleotide primers. The method may also use each set of primers in step (f) are used in a separate amplification. Furthermore, the 5′ sequence of one or both of the primer sequences in step (f) may comprise a recognition sequence for a restriction enzyme. In other preferred embodiments, at least one of the primers in step (f) is labeled, such as with a fluorescent label.
In another preferred embodiment, 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. Moreover, in certain embodiments, one of the two strands of the noncomplementary portion comprises a recognition sequence for a restriction enzyme.
In related aspects, the present invention provides a method for selectively isolating in a nucleic acid sample DNA fragments having sequences corresponding to 3′ ends of mRNAs, comprising the steps above plus isolating the amplified fragment; cloning the isolated fragment, such as by digesting the amplified fragments in step (f) with a restriction enzyme, and ligating the digested fragments to a vector, and further determining the DNA sequence of the isolated fragment; isolating and analyzing the amplified fragment (e.g., determining the DNA sequence, hybridizing to nucleic acid molecules); and detecting the amplified fragments (e.g., by hybridizing the fragments to nucleic acid molecules.
In preferred embodiments, the nucleic acid molecules are attached to a silicon wafer or porous glass wafer, the nucleic acid molecules are oligonucleotides from about 25 to about 40 nucleotides long, the nucleic acid molecules comprise a set of cDNA sequences, and/or the fragments are labeled.
In yet another aspect, the invention provides 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 and comparing the amounts of amplified fragments. In preferred embodiments, one of the cell populations is treated or is a tumor cell population.
The invention also provides 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, wherei
Prashar Yatindra
Weissman Sherman M.
Horlick Kenneth R.
Morgan & Lewis & Bockius, LLP
Tung J.
Yale University
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