Processes for genetic manipulations using promoters
DCFirst Claim
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1. A process for producing a subtractive hybridization probe comprising:
- (a) synthesizing a first double-stranded cDNA collection by treating a first mRNA population with a primer complex, wherein the primer is complementary to the RNA sequence and is operably linked to a first promoter region for transcription of the cDNA strand complementary to the primer;
(b) transcribing the first cDNA into anti-sense RNA by introducing a first RNA polymerase that binds to the first promoter region;
(c) hybridizing the anti-sense RNA to a second mRNA population, whereby an unhybridized subpopulation of the second RNA population is separated;
(d) generating a second double-stranded cDNA collection from the unhybridized subpopulation using a second primer complex comprising a second promoter region in an orientation for transcribing anti-sense RNA complementary to the unhybridized subpopulation; and
(e) transcribing the second cDNA into a ribonucleotide probe by introducing a second RNA polymerase that binds to the second promoter region and transcribing RNA which is complimentary to the mRNA in the unhybridized subpopulation of the second RNA population.
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Abstract
This invention relates to the use of promoters for ribonucleic acid amplification and other genetic manipulations. Processes are provided wherein complementary deoxyribonucleic acid (cDNA) is synthesized from a ribonucleic acid (RNA) sequence using a complementary primer linked to an RNA polymerase promoter region complement and then anti-sense RNA (aRNA) is transcribed from the cDNA by introducing an RNA polymerase capable of binding to the promoter region. Additional processes using the resulting aRNA are also described.
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Citations
42 Claims
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1. A process for producing a subtractive hybridization probe comprising:
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(a) synthesizing a first double-stranded cDNA collection by treating a first mRNA population with a primer complex, wherein the primer is complementary to the RNA sequence and is operably linked to a first promoter region for transcription of the cDNA strand complementary to the primer; (b) transcribing the first cDNA into anti-sense RNA by introducing a first RNA polymerase that binds to the first promoter region; (c) hybridizing the anti-sense RNA to a second mRNA population, whereby an unhybridized subpopulation of the second RNA population is separated; (d) generating a second double-stranded cDNA collection from the unhybridized subpopulation using a second primer complex comprising a second promoter region in an orientation for transcribing anti-sense RNA complementary to the unhybridized subpopulation; and (e) transcribing the second cDNA into a ribonucleotide probe by introducing a second RNA polymerase that binds to the second promoter region and transcribing RNA which is complimentary to the mRNA in the unhybridized subpopulation of the second RNA population.
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2. A method for making a cDNA library from a collection of mRNA molecules comprising:
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(a) hybridizing one or more primer complexes to a plurality of the mRNA'"'"'s wherein each complex comprises an oligonucleotide primer linked to a promoter sequence; (b) producing a collection of double-stranded cDNAs by extending the primers of a plurality of any hybridization duplexes formed between the mRNAs and the complexes wherein each double stranded cDNA comprises a first cDNA strand which is complementary to one mRNA molecule and operably linked to the promoter, and a second strand which is complementary to the first cDNA strand operably linked to the promoter; (c) transcribing multiple copies of anti-sense RNA off of the second strand; and (d) preparing a cDNA library from the anti-sense RNA copies.
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3. A process for amplifying at least one target nucleic acid sequence comprising:
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(a) synthesizing a double-stranded nucleic acid by; i) hybridizing a primer complex to the target nucleic acid sequence and extending the primer complex to form a first DNA strand complementary to the target sequence, wherein said primer complex comprises a promoter and a primer region complementary to the target nucleic acid sequence, and ii) synthesizing a second DNA strand complementary to the first DNA strand without using an exogenous primer complementary to the first DNA strand; and
,(b) transcribing copies of RNA initiated from the promoter region of the primer complex, wherein said copies of RNA are complementary to the second DNA strand. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A process for detecting expression of at least one gene in a preselected cell population comprising:
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(a) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by; (i) hybridizing a primer complex comprising a promoter and a polythymidylate (poly(dT)) region complementary to mRNA present in the cell population, (ii) extending the primer complex to form a first cDNA strand, and, (iii) synthesizing a second cDNA strand complementary to the first cDNA strand without using an exogenous primer complementary to the first cDNA strand; (b) transcribing the double-stranded cDNA into anti-sense RNA (aRNA); and
,(c) determining the presence of aRNA corresponding to the gene or genes, wherein the presence of aRNA complementary to a gene corresponds to expression of a gene. - View Dependent Claims (15)
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16. A method for subtractive hybridization comprising the steps:
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(a) binding a primer to sense RNA molecules in a first population, wherein the primer is operably linked to a promoter sequence in an anti-sense orientation; (b) synthesizing a first complementary DNA (cDNA) strand by elongation from the primer; (c) synthesizing a second cDNA strand without using an exogenous primer complementary to the first strand, whereupon a functional promoter is generated; (d) initiating RNA synthesis from the promoter by adding RNA polymerase, whereby anti-sense RNA (aRNA) is produced; (e) introducing the aRNA in molar excess to a second population of sense RNA molecules, whereby complementary RNA sequences from the two populations hybridize; and (f) isolating remaining single-stranded sense RNA. - View Dependent Claims (17)
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18. A method for detecting the expression in one or more cells of at least one gene, said method comprising the steps:
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(a) hybridizing a primer complex to RNA molecules in a population of RNA molecules from said cell, wherein the primer complex comprises a promoter sequence and a primer sequence complementary to at least one RNA in the population; (b) synthesizing a first complementary DNA (cDNA) strand by elongation from said primer; (c) synthesizing a second complementary cDNA strand without using an exogenous primer complementary to the first cDNA strand, wherein a functional promoter is generated; (d) initiating RNA synthesis from the promoter by adding RNA polymerase, thereby producing amplified antisense RNA (aRNA); and (e) detecting the presence of aRNA complementary to the mRNA transcribed from the gene and relating the presence of said aRNA to the expression of the gene in the cell. - View Dependent Claims (19, 20, 21, 22, 23)
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24. A method for amplifying a brain cell mRNA comprising:
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(a) introducing into a brain cell a reaction mixture comprising; (I) a primer complex operably bound to a promoter sequence in the anti-sense orientation, ii) reverse transcriptase, and iii) dATP, dCTP, dGTP, and dTTP, thereby generating a first strand cDNA; (b) harvesting the brain cell; (c) precipitating nucleic acids from the brain cell and recovering the first strand cDNA; (d) synthesizing second-strand cDNA without using an exogenous primer complementary to the first cDNA strand; (e) initiating transcription by adding RNA polymerase, rATP, rCTP, rGTP and UTP; (f) whereby anti-sense RNA is produced and a brain cell mRNA is amplified.
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25. A process for determining the level of expression of at least one mRNAs in a preselected cell or cell population relative to the level of expression of the other mRNAs in the same cell or cell population comprising:
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(a) adding a primer complex to a mixture comprising a population of mRNAs from said preselected cell or cell population, said primer complex comprising i) a primer sequence complementary to a plurality of said mRNAs, and ii) a promoter sequence in antisense orientation; whereupon the primer complex hybridizes to said plurality of mRNAs; (b) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by i) extending the hybridized primer complexes to produce first strand cDNAs, ii) synthesizing second strand cDNAs without using an exogenous primer, wherein the second strand comprises the promoter sequence in sense orientation; (c) transcribing multiple copies of antisense RNA (aRNA) initiated from the promoter region of the primer complexes to produce a population of aRNAs; and
,(d) analyzing the population of aRNAs to determine the level of representation of a specific aRNA sequence relative to other aRNA sequences within the population of aRNAs, wherein the amount of a specific aRNA in a population of aRNAs corresponds to the amount of the corresponding mRNA in the population of mRNAs. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33)
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34. A process for comparing mRNA expression in different tissues or different physiologic states of the same tissue, said process comprising:
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(a) adding a primer complex to a mixture comprising a population of mRNAs from a first cell population, said primer complex comprising i) a primer sequence complementary to a plurality of said mRNAs, and ii) a promoter sequence in antisense orientation; whereupon the primer complex hybridizes to said plurality of mRNAs; (b) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by i) extending the hybridized primer complexes to produce first strand cDNAs, ii) synthesizing second strand cDNAs without using an exogenous primer, wherein the second strand comprises the promoter sequence in sense orientation; (c) transcribing multiple copies of amplified RNA initiated from the promoter region of the primer complexes to produce a population of amplified RNAs; (d) carrying out steps (a)-(c) by adding the primer complex of step (a) to a mixture comprising a population of mRNAs from a second preselected cell population, wherein said first and second cell populations are from different tissues or different physiologic states of the same tissue; and
,(e) comparing the species of amplified RNA produced from the first cell population to the to same species of amplified RNA produced from the second cell population, wherein the amount of the amplified RNA in each population of amplified RNAs corresponds to the amount of the corresponding mRNA in the corresponding cell population. - View Dependent Claims (35, 36, 37, 38)
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39. A component kit for the production of amplified RNA comprising:
- a container comprising a primer complex component and instructions for use of said primer complex wherein said primer complex component comprises a polythymidylate (poly(dT)) region and a promoter.
- View Dependent Claims (40, 41)
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42. A component kit for the production of amplified RNA comprising:
- a container comprising a plurality of primer complex species, wherein said primer complex species comprise random primers linked to a promoter, and instructions for use of said primer complex species.
Specification