Claims
- 1. A method of screening a population of nucleic acids for a novel sequence, the method comprising:
providing a population of nucleic acid sequences; partitioning said population into one or more subpopulations of nucleic acids; identifying a first nucleic acid sequence in the subpopulation of nucleic acid sequences; and comparing the first nucleic acid sequence to a reference nucleic acid sequence or sequences, wherein the absence of the first nucleic acid sequence in the reference nucleic acid or nucleic acid sequences indicates the first nucleic acid is a novel nucleic acid sequence.
- 2. The method of claim 1, wherein said DNA population is a cDNA population derived from a population of RNA molecules.
- 3. The method of claim 2, further comprising partitioning the RNA molecules.
- 4. The method of claim 2, wherein said cDNA population is derived from the 5′ ends of the RNA molecules.
- 5. The method of claim 2, wherein said cDNA population is derived from the interior regions of the RNA molecules.
- 6. The method of claim 2, wherein said cDNA population is derived from the 3′ ends of the DNA molecules.
- 7. The method of claim 2, wherein said partitioning step comprises hybridization of a probe nucleic acid sequence to the population of nucleic acids.
- 8. The method of claim 2, wherein said partitioning step comprises digesting the cDNA molecules with one or more restriction enzymes.
- 9. The method of claim 8, further comprising ligating adapter oligonucleotides to the termini of the digested cDNA molecules.
- 10. The method of claim 9, further comprising amplifying the ligation products.
- 11. The method of claim 8, further comprising separating the amplified products.
- 12. The method of claim 11, wherein said separating is by gel electrophoresis.
- 13. The method of claim 11, wherein the first nucleic acid sequence is identified by comparing the size of one or more digestion products produced by a member of the subpopulation of nucleic acids to the sizes of fragments generated by the same restriction enzyme or enzymes in said reference nucleic acid or nucleic acids.
- 14. The method of claim 11, further comprising
recovering one or more size-separated digestion products; reamplifying the recovered products; and separating the reamplified products.
- 15. The method of claim 14, wherein said separating is by gel electrophoresis.
- 16. The method of claim 15, wherein the first nucleic acid sequence is identified by comparing the size of one or more digestion products produced by a member of the subpopulation of nucleic acids to the sizes of fragments generated by the same restriction enzyme or enzymes in said reference nucleic acid or nucleic acids.
- 17. The method of claim 9, further comprising:
inserting the ligated adapter oligonucleotide into a cloning vector to form a vector-insert; transforming the vector-insert into a suitable host; culturing transformed host under conditions allowing for replication of the vector-insert; recovering the vector-insert from said host; and digesting the vector-insert with one or more restriction enzymes, thereby releasing said insert; and comparing the size of the insert to sizes of fragments generated by the same restriction enzyme or enzymes in said reference nucleic acid or nucleic acids.
- 18. The method of claim 1, wherein comparing is by determining at least a portion of the nucleotide sequence of the first nucleic acid sequence and comparing the nucleotide sequence to the nucleotide sequence of one or more reference nucleic acids.
- 19. The method of claim 1, wherein comparing is by hybridizing the first nucleic acid sequence to one or more of the reference nucleic acid sequences.
- 20. A method for equalizing the representation of nucleic acids in a population of nucleic acids, the method comprising:
providing a population of nucleic acid sequences, wherein said population comprises a first nucleic acid and a second nucleic acid having a nucleic acid sequence distinct from the first nucleic acid, and wherein said first nucleic acid is present at a higher level in said population than said second population; partitioning said population into one or more subpopulations of nucleic acids; and comparing the levels of said first nucleic acid sequence to the levels of said second nucleic acid sequence in the subpopulation of nucleic acid sequences, wherein a lower level of the first nucleic acid sequence relative to the second nucleic acid sequence indicates the representation of said first and second nucleic acid sequences are normalized.
- 21. A method for producing a population of nucleic acid molecules enriched for 5′ regions of mRNA molecules, the method comprising:
providing a population of RNA molecules, said population including RNA molecules having a 5′ terminal Gppp cap structure and a 5′ terminal phosphate group; contacting said population of RNA molecules with a phosphatase under conditions that result in removal of the 5′ terminal phosphate group while leaving the 5′ terminal Gppp cap structure intact; inactivating said phosphatase; contacting the population of RNA molecules with a pyrophosphatase under conditions that result in the removal of the 5′ terminal Gppp and the formation of a 5′ phosphate group; annealing an oligonucleotide in the presence of an RNA ligase to form a hybrid molecule; and forming a cDNA from said oligonucleotide.
- 22. A method of identifying an RNA sequence in a sample comprising a plurality of RNA sequences, the method comprising:
synthesizing cDNA copies of a plurality of RNA species to form a cDNA sample; determining the size of one or more of said cDNA molecules in said cDNA sample; comparing the size of said sample with the size of a reference nucleic acid; and thereby identifying the cDNA sequence.
- 23. The method of claim 22, wherein said cDNA molecules are digested with one or more restriction enzymes prior to the determining step.
- 24. The method of claim 23, further comprising ligating adapter oligonucleotides to the termini of the digested cDNA molecules prior to the determining step.
- 25. The method of claim 22, wherein said identifying step comprises comparing the size of one or more digestion products produced by one or more said cDNA molecules to a reference nucleic acid or nucleic acids.
- 26. A method of identifying an RNA sequence in a population of RNA sequences, the method comprising:
(a) removing 5′ terminal pppG from RNAs in said population to form a population of RNAs having terminal 5′ phosphate groups; (b) ligating a linker oligonucleotide to the terminal 5′ phosphate groups of RNA molecules in said population of RNAs; (c) synthesizing complementary cDNA molecules from said population of RNA molecules to form a cDNA sample; (d) digesting said complementary cDNA molecules with at least one restriction enzyme; (e) ligating an adapter molecule to the digested cDNA molecules; (f) amplifying the molecules produced in step (e); (g) identifying the amplified molecules of step (f); and (h) comparing the amplified molecules to one or more reference nucleic acids.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No. 60/115,109, filed Jan. 8, 1999, which is incorporated herein in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60115109 |
Jan 1999 |
US |