CA2294962A1 - Nucleic acid sequence analysis - Google Patents
Nucleic acid sequence analysis Download PDFInfo
- Publication number
- CA2294962A1 CA2294962A1 CA002294962A CA2294962A CA2294962A1 CA 2294962 A1 CA2294962 A1 CA 2294962A1 CA 002294962 A CA002294962 A CA 002294962A CA 2294962 A CA2294962 A CA 2294962A CA 2294962 A1 CA2294962 A1 CA 2294962A1
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- Prior art keywords
- group
- blocking group
- nucleotide
- polymerase
- blocking
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention relates to a method for determining the sequence of a polynucleotide, the method comprising the steps of: (i) reacting a target polynucleotide with a polymerase enzyme immobilised on a solid support, and the different nucleotides, under conditions sufficient for the polymerase reaction; and (ii) detecting the incorporation of a specific nucleotide complementary to the target polynucleotide, by measuring radiation.
Claims (29)
1. A method for sequencing a polynucleotide, comprising the steps of:
(i) reacting a target polynucleotide with a polymerase enzyme immobilised on a solid support, and the different nucleotides, under conditions sufficient for the polymerase reaction; and (ii) detecting an effect consequent on the incorporation of a specific nucleotide complementary to the target polynucleotide.
(i) reacting a target polynucleotide with a polymerase enzyme immobilised on a solid support, and the different nucleotides, under conditions sufficient for the polymerase reaction; and (ii) detecting an effect consequent on the incorporation of a specific nucleotide complementary to the target polynucleotide.
2. A method according to claim 1, wherein the effect in step (ii) is detected by measuring radiation.
3. A method according to claim 1 or claim 2, wherein steps (i) and (ii) are conducted with each of the different nucleotides in turn, until incorporation is detected, and then repeated.
4. A method according to claim 1 or claim 2, wherein step (i) is conducted with all the nucleotides present.
5. A method according to any preceding claim, wherein the nucleotides comprise a 3' blocking group which is removed after the polymerase reaction.
6. A method according to claim 5, wherein the blocking group can be selectively removed by pulsed monochromatic light.
7. A method according to claim 5 or claim 6, wherein the nucleotides comprise a further blocking group at the terminal phosphate group of the triphosphate chain, and the further blocking group is removed prior to the removal of the 3' blocking group.
8. A method according to claim 7, wherein the further blocking group can be selectively removed by pulsed monochromatic light under conditions different from those required to remove the 3' blocking group.
9. A method according to claim 8, wherein the further blocking group is removed by pulsing the monochromatic light for a duration different from that required to remove the 3' blocking group.
10. A method according to any preceding claim, wherein step (i) further comprises introducing a competitive inhibitor of the polymerise enzyme.
11. A method according to any preceding claim, wherein the target polynucleotide of step (i) is bound to the polymerise enzyme by a .beta.2 dimer complex.
12. A method according to any preceding claim, wherein the polymerise is E. coli DNA polymerise III or T7 polymerase.
13. A method according to any of claims 1 to 11, wherein the polymerase is Taq polymerase.
14. A method according to any of claims 1 to 11, wherein the polymerase is reverse transcriptase.
15. A method according to any preceding claim, wherein step (ii) comprises detection of a change in resonance signal over time.
16. A method according to any preceding claim, wherein the radiation is electromagnetic.
17. A method according to claim 16, wherein the electromagnetic radiation is in the infra-red spectrum.
18. A method according to any preceding claim, wherein step (ii) comprises using surface plasmon resonance.
19. A method according to claim 16, wherein the electromagnetic radiation is in the radio-frequency spectrum.
20. A method according to claim 19, wherein the incorporation of a nucleotide is detected using NMR.
21. A method according to any preceding claim, wherein the polynucleotide is DNA.
22. A sensor chip comprising a polymerase enzyme immobilised thereon.
23. A nucleotide comprising a blocking group at the 3' position and at the terminal phosphate group of the triphosphate chain, wherein the two blocking groups are removable by monochromatic light of different wavelengths.
24. A nucleotide according to claim 23, wherein the blocking groups are derived from a compound of the formula R1-[O-CO-]X
wherein R1 is a photolabile group and X is a leaving group.
wherein R1 is a photolabile group and X is a leaving group.
25. A nucleotide according to claim 23 or claim 24, wherein the blocking group at the 3' position is an o-nitrobenzyloxycarbonyl group.
26. A nucleotide according to any of claims 23 to 25, wherein the blocking group at the terminal phosphate is an o-nitrobenzyl group.
27. A nucleotide according to any of claims 23 to 26, wherein the blocking group at the 3' position is a (4,5-dimethoxy-2-nitrobenzyl)oxycarbonyl group.
28. A nucleotide according to any of claims 23 to 27, wherein the blocking group at the terminal phosphate is a 1-(2-nitrophenyl)ethyl group.
29. An apparatus for sequencing a polynucleotide, comprising an optical sensor chip, a light source, an imaging device and a photodetector, wherein the sensor chip is as defined in claim 22.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9715942.0A GB9715942D0 (en) | 1997-07-28 | 1997-07-28 | |
GB9715942.0 | 1997-07-28 | ||
GB9727103.5 | 1997-12-22 | ||
GBGB9727103.5A GB9727103D0 (en) | 1997-12-22 | 1997-12-22 | |
PCT/GB1998/002214 WO1999005315A2 (en) | 1997-07-28 | 1998-07-24 | Nucleic acid sequence analysis |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2294962A1 true CA2294962A1 (en) | 1999-02-04 |
CA2294962C CA2294962C (en) | 2012-09-18 |
Family
ID=26311958
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2294962A Expired - Lifetime CA2294962C (en) | 1997-07-28 | 1998-07-24 | Nucleic acid sequence analysis |
Country Status (19)
Country | Link |
---|---|
US (4) | US7008766B1 (en) |
EP (4) | EP2267164B1 (en) |
JP (1) | JP2001511358A (en) |
KR (1) | KR100664331B1 (en) |
CN (1) | CN1152140C (en) |
AT (1) | ATE225858T1 (en) |
AU (1) | AU735898B2 (en) |
BR (1) | BR9812270A (en) |
CA (1) | CA2294962C (en) |
DE (1) | DE69808661T3 (en) |
DK (1) | DK1017848T3 (en) |
ES (1) | ES2183394T5 (en) |
HK (1) | HK1031130A1 (en) |
IL (1) | IL133411A (en) |
IS (1) | IS5360A (en) |
NZ (1) | NZ502557A (en) |
PT (1) | PT1017848E (en) |
RU (1) | RU2198221C2 (en) |
WO (1) | WO1999005315A2 (en) |
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