US5374527A
Expired / Lapsed
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High resolution DNA sequencing method using low viscosity medium
Assignee: Applied Biosystems LLC
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Filing: 1993-01-21
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Granted: 1994-12-20
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Expires: 2013-01-21
Abstract
Abstract A DNA sequencing method for use in sequencing a DNA target sequence up to 300 bases, preferably up to 500 bases or greater in length, by electrophoretically separating a mixture of single-stranded DNA sequencing fragments in a capillary tube. The method employs an aqueous denaturing solution comprising between about 4 and about 7 weight percent linear polyacrylamide molecules having an average molecular weight of between about 20 and about 100 kDa. The low-viscosity of the solution allows rapid loading and reloading of such solution into the capillary tube.
Inventors
- 👤 Paul D. Grossman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 09:37
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
60 words
In a method of determining the nucleotide sequence of a target polynucleotide, wherein DNA fragments in a DNA sequencing mixture are separated by size, the improvement comprising: separating said DNA fragments by capillary electrophoresis in an aqueous denaturing solution comprising between 4 and 7 weight percent non-crosslinked polyacrylamide molecules having an average molecular weight of between 20 and 100 kilodaltons.
Claim 2
Dependent Claim
60 words
In a method of determining the nucleotide sequence of a target polynucleotide, wherein DNA fragments in a DNA sequencing mixture are separated by size, the improvement comprising: separating said DNA fragments by capillary electrophoresis in an aqueous denaturing solution comprising between 4 and 7 weight percent non-crosslinked polyacrylamide molecules having an average molecular weight of between 20 and 100 kilodaltons.
Claim 3
Dependent Claim
12 words
The method of claim 1, wherein said polyacrylamide molecules are linear polymers.
Claim 4
Dependent Claim
14 words
The method of claim 2, wherein said average molecular weight is about 55 kilodaltons.
Claim 5
Dependent Claim
17 words
The method of claim 1, which is carried out in parallel with a plurality of capillary tubes.
Claim 6
Dependent Claim
23 words
The method of claim 1, wherein by said separating, a single-base resolution is achieved for fragments of at least 300 bases in length.
Claim 7
Dependent Claim
23 words
The method of claim 1, wherein by said separating, a single-base resolution is achieved for fragments of at least 500 bases in length.
Claim 8
Dependent Claim
26 words
The method of claim 1, wherein said mixture includes two distinguishable reporters by which the 3'-terminal base in a plurality of said fragments can be identified.
Claim 9
Dependent Claim
25 words
The method of claim 1, wherein said mixture includes four distinguishable reporters for use in identifying the 3'-terminal base in a plurality of said fragments.
Claim 10
Dependent Claim
10 words
The method of claim 8, wherein said reporters are fluorescent.
Claim 11
Dependent Claim
45 words
The method of claim 1, wherein after said separating has been carried out in a capillary tube, said aqueous denaturing solution in said capillary tube is replaced with fresh aqueous denaturing solution, whereby said capillary tube is now usable for separating another polynucleotide fragment sample.
Claim 12
Dependent Claim
108 words
In a method of determining the nucleotide sequence of a target polynucleotide, which includes separating DNA fragments in a DNA sequencing mixture by size in a capillary electrophoresis tube, the improvement comprising the steps of: (a) separating said DNA fragments by capillary electrophoresis in an aqueous solution effective to denature double stranded DNA and comprising between 4 and 7 weight percent non-crosslinked polyacrylamide molecules having an average molecular weight of between 20 and 100 kilodaltons, and (b) after said separating, removing said solution from said tube and introducing fresh aqueous denaturing solution comprising non-crosslinked polyacrylamide molecules in said tube by applying pressure to one end of said tube.
Claim 13
Dependent Claim
108 words
In a method of determining the nucleotide sequence of a target polynucleotide, which includes separating DNA fragments in a DNA sequencing mixture by size in a capillary electrophoresis tube, the improvement comprising the steps of: (a) separating said DNA fragments by capillary electrophoresis in an aqueous solution effective to denature double stranded DNA and comprising between 4 and 7 weight percent non-crosslinked polyacrylamide molecules having an average molecular weight of between 20 and 100 kilodaltons, and (b) after said separating, removing said solution from said tube and introducing fresh aqueous denaturing solution comprising non-crosslinked polyacrylamide molecules in said tube by applying pressure to one end of said tube.
Claim 14
Dependent Claim
13 words
The method of claim 11, wherein said polyacrylamide molecules are linear polyacrylamide molecules.
Claim 15
Dependent Claim
14 words
The method of claim 12, wherein said average molecular weight is about 55 kilodaltons.
Claim 16
Dependent Claim
20 words
The method of claim 11, wherein the solution has a viscosity of between 10 and 300 centipoise at 30° C.
Claim 17
Dependent Claim
24 words
The method of claim 11, wherein by said separating, a single-base resolution is achieved for fragments of at least about 300 bases in length.
Claim 18
Dependent Claim
23 words
The method of claim 11, wherein by said separating, a single-base resolution is achieved for fragments of at least 500 bases in length.
Claim 19
Dependent Claim
15 words
The method of claim 11, carried out in parallel with a plurality of capillary tubes.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1997-12-19 | 1998-06-20 | 1998-12-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-12-19 | 2002-06-20 | 2002-12-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-12-19 | 2006-06-20 | 2006-12-20 | $7700.00 | Window Closed / Lapsed |