Method of obtaining a library of tags capable of defining a specific state of a biological sample
First Claim
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1. A method of obtained a library of tags capable of defining a specific state of a biological sample, comprising:
- (1) extracting mRNA from a biological sample comprising ≦
5×
106 cells, corresponding to at most 50 μ
g of total RNA or 1 μ
g of poly(A) RNA, by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads, (2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;
(a) synthesizing the 1st strand of the cDNA by reverse transcription of the mRNA template into a 1st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and (b) synthesizing the 2nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an E. coli DNA polymerase, (3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site two different hybrid DNA molecules composed from linkers 1A and 1B or from linkers 2A and 2B, wherein the linkers have the following formulas;
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Abstract
A method of obtaining a library of tags able to define a specific state of a biological sample, such as a tissue or a cell culture. The present method provides an important advantage over other methods used to analyze gene expression in that libraries may be generated from tiny amounts of cells, e.g., from 30,000-100,000 cells.
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Citations
19 Claims
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1. A method of obtained a library of tags capable of defining a specific state of a biological sample, comprising:
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(1) extracting mRNA from a biological sample comprising ≦
5×
106 cells, corresponding to at most 50 μ
g of total RNA or 1 μ
g of poly(A) RNA, by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,(2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;
(a) synthesizing the 1st strand of the cDNA by reverse transcription of the mRNA template into a 1st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and (b) synthesizing the 2nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an E. coli DNA polymerase, (3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site two different hybrid DNA molecules composed from linkers 1A and 1B or from linkers 2A and 2B, wherein the linkers have the following formulas;
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of obtaining a library of tags able to define a specific state of biological sample, comprising:
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(1) extracting mRNA from biological sample comprising ≦
5×
106 cells, corresponding to at most 50 μ
g of total RNA or 1 μ
g of poly(A) RNA, by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,(2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;
(a) synthesizing the 1st strand of the cDNA by reverse transcription of the mRNA template into a 1st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and (b) synthesizing a 2nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid-form by an E. coli DNA polymerase, (3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site to a linker consisting of one double-strand cDNA molecule composed from linkers 1A and 1B or from linkers 2A and 2B. wherein the linkers have the following formulas;
- View Dependent Claims (14, 15, 16, 17, 18, 19)
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Specification