EP0781411A1 - Trennung von nukleinsäuren durch kapillarelektroforese mit thermischen gradienten in viskosen polymerlösungen - Google Patents
Trennung von nukleinsäuren durch kapillarelektroforese mit thermischen gradienten in viskosen polymerlösungenInfo
- Publication number
- EP0781411A1 EP0781411A1 EP95932707A EP95932707A EP0781411A1 EP 0781411 A1 EP0781411 A1 EP 0781411A1 EP 95932707 A EP95932707 A EP 95932707A EP 95932707 A EP95932707 A EP 95932707A EP 0781411 A1 EP0781411 A1 EP 0781411A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- separation
- capillary
- process according
- dna
- temperature
- 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.)
- Withdrawn
Links
- 238000000926 separation method Methods 0.000 title claims abstract description 45
- 229920000642 polymer Polymers 0.000 title claims abstract description 30
- 150000007523 nucleic acids Chemical class 0.000 title claims description 7
- 238000005251 capillar electrophoresis Methods 0.000 title claims description 6
- 108020004707 nucleic acids Proteins 0.000 title description 5
- 102000039446 nucleic acids Human genes 0.000 title description 5
- 238000000034 method Methods 0.000 claims abstract description 33
- 229920002401 polyacrylamide Polymers 0.000 claims abstract description 30
- 239000012634 fragment Substances 0.000 claims abstract description 22
- 230000035772 mutation Effects 0.000 claims abstract description 15
- 239000000178 monomer Substances 0.000 claims abstract description 5
- 230000002123 temporal effect Effects 0.000 claims abstract description 5
- PNAFOQXNEZSHNR-UHFFFAOYSA-N n-(1-ethoxy-1-hydroxyethyl)prop-2-enamide Chemical compound CCOC(C)(O)NC(=O)C=C PNAFOQXNEZSHNR-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000008569 process Effects 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 238000002844 melting Methods 0.000 claims description 9
- 230000008018 melting Effects 0.000 claims description 9
- 238000012546 transfer Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 238000007873 sieving Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000013508 migration Methods 0.000 claims description 4
- 230000005012 migration Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229920000609 methyl cellulose Polymers 0.000 claims description 3
- 235000010981 methylcellulose Nutrition 0.000 claims description 3
- 229920000936 Agarose Polymers 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims description 2
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 claims description 2
- 239000004354 Hydroxyethyl cellulose Substances 0.000 claims description 2
- -1 N-substituted acrylamides Chemical class 0.000 claims description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- 230000017525 heat dissipation Effects 0.000 claims description 2
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 claims description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 claims description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 claims description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 claims description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 2
- 239000001923 methylcellulose Substances 0.000 claims description 2
- 238000005457 optimization Methods 0.000 claims description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 2
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 claims 1
- 229920002581 Glucomannan Polymers 0.000 claims 1
- 239000004373 Pullulan Substances 0.000 claims 1
- 229920001218 Pullulan Polymers 0.000 claims 1
- 125000004122 cyclic group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 238000001917 fluorescence detection Methods 0.000 claims 1
- 238000005194 fractionation Methods 0.000 claims 1
- 229940046240 glucomannan Drugs 0.000 claims 1
- 238000010438 heat treatment Methods 0.000 claims 1
- 235000019423 pullulan Nutrition 0.000 claims 1
- 238000005515 capillary zone electrophoresis Methods 0.000 abstract description 11
- 238000001499 laser induced fluorescence spectroscopy Methods 0.000 abstract description 3
- YPHQUSNPXDGUHL-UHFFFAOYSA-N n-methylprop-2-enamide Chemical compound CNC(=O)C=C YPHQUSNPXDGUHL-UHFFFAOYSA-N 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 31
- 238000004458 analytical method Methods 0.000 description 9
- 239000003398 denaturant Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 239000004202 carbamide Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000004907 flux Effects 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 108700024394 Exon Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 101150029409 CFTR gene Proteins 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000379 polymerizing effect Effects 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000000518 rheometry Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000004642 Polyimide Substances 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 241000944748 Quesada Species 0.000 description 1
- 229920001938 Vegetable gum Polymers 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000018984 mastication Effects 0.000 description 1
- 238000010077 mastication Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920001721 polyimide Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
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- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44734—Arrangements for investigating the separated zones, e.g. localising zones by thermal means
Definitions
- FIG. 1 shows the separation of an amplified DNA fragment (cystic fibrosis, CF, gene from a normal individual) in the absence (lower tracing) and presence (upper profile) of thermal denaturing gradients. In the lower tracing, separation occurs at constant temperature (45 * C) and in presence of chemical denaturants (6 M urea). The peaks eluted between 27 and 35 min represent oligonucleotide primers. The normal amplified DNA is eluted as a single peak (labelled W t /W t ) between 58 and 60 min.
- Fig. 3 shows the analysis of a set of intermediate melting fragments, amplified from CF patients heterozygous for different mutations in exon 11 of the CFTR gene: 1717-1G —> A (panel A); G542X (G —> T at 1756; panel C) and 1784delG (panel D) with their respective normal control (panel E). All mutants exhibit the characteristic four-peak profile, vs. a single band in the control. As shown in the temperature profile of panel E, these mutants are intermediate melters, with T ⁇ 's in the 56.5 to 57.8*C range.
- Fig. 4 shows the optimized condition set up for a higher melting fragment, amplified from a CF patient homozygous for the M1V mutation (A --> G transversion at position 133 in exon 1 of the CFTR gene).
- the panel shows the electropherogram of the sample injected at a constant temperature plateau (65'C), constant denaturant buffer, but in the absence of a temperature gradient.
- 65'C constant temperature plateau
- the group of peaks eluting from 35 to 48 min corresponds to unpurified primers with an without GC-cla ps.
- the insert shows the optimized separation in a 65 to 67 * C gradient with a slope of O.l'C/min: the correct spectrum of four bands is now obtained.
- the temperature is the one truly existing inside the capillary and is precisely determined with the aid of computer programs developed by us (M.S. Bello, E.I. Levine & P.O. Righetti: Computer assisted determination of the inner temperature and peak correction for capillary zone electrophoresis. J. Chromatogr. 652, 1993, 329-336). Use of masticated and "chain-transfer" polyacrylamides.
- Fig. 5 shows the progressive decrements of viscosity and average molecular mass of polyacrylamides during the mastication process by sonication.
- the viscosity has been measured with a Bohlin VOR rheo eter (Bohlin Rheology, Lund, Sweden), whereas M has been determined by gel permeation (HPLC Waters' 590 Solvent Delivery System, equipped with two Waters Ultrahydrogel columns and with a differential refractometric detector R401 against polyethylene glycol standards.
- Such reduced viscosity allows the injection into the capillary of much more concentrated solutions of polyacrylamides (up to 10%), which in turn permit optimization of resolution in the DNA size interval (typically from 100 to 500 bp) most interesting for the screening of genetic mutations via analysis of amplified DNA fragments.
- Even better separations can be achieved by polymerization in presence of chain-transfer agents (e.g., 3% isopropanol) coupled to elevated temperatures, a process which generates chains of further reduced lengths and viscosities.
- Fig. 5 shows viscosity measurements as a function of polymer concentration obtained by polymerization in presence of "chain transfer" agents at 35 * C and at 70 * C.
- the viscosity has been measured with a Bohlin VOR rheometer (Bohlin Rheology, Lund, Swden).
- Bohlin Rheology Lund, Swden
- the drastic viscosity reduction at high temperatures is due to formation of short chains (M r of only 180000 Da at 70 * C, as opposed to M r cf 450000 Da when polymerizing at 35'C).
- the viscosities of polyacrylamides polymerized at 35 or at 70 * C are markedly different.
- a strong decrement of viscosity is obtained (e.g., in an 8% polymer solution, the viscosity diminishes from 450 mPa.s to barely 120 mPa.s).
- This strong viscosity decrement is due to a marked reduction in average chain length, which diminishes from 430000 Da (when polymerizing at 35 * C) to only 180000 Da at 70 * C.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITMI941864A IT1271005B (it) | 1994-09-12 | 1994-09-12 | Separazione di acidi nucleici per elettroforesi capillare in gradienti termici e in soluzioni viscose di polimeri |
| ITMI941864 | 1994-09-12 | ||
| PCT/EP1995/003561 WO1996008715A1 (en) | 1994-09-12 | 1995-09-11 | Separation of nucleic acids by capillary electrophoresis in thermal gradients in viscous polymer solutions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0781411A1 true EP0781411A1 (de) | 1997-07-02 |
Family
ID=11369544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP95932707A Withdrawn EP0781411A1 (de) | 1994-09-12 | 1995-09-11 | Trennung von nukleinsäuren durch kapillarelektroforese mit thermischen gradienten in viskosen polymerlösungen |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0781411A1 (de) |
| JP (1) | JP3015106B2 (de) |
| AU (1) | AU689648B2 (de) |
| IT (1) | IT1271005B (de) |
| WO (1) | WO1996008715A1 (de) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9912743D0 (en) * | 1999-06-02 | 1999-08-04 | Proteome Sciences Plc | Method and kit for ligand assay |
| EP1285260A4 (de) * | 2000-04-25 | 2006-02-01 | Spectrumedix Llc | Denaturiermittelfreie elektrophorese biologischer moleküle bei hohen temperaturen |
| EP1320745A4 (de) | 2000-09-01 | 2008-10-01 | Applera Corp | System und verfahren zur temperaturgradienten-kapillarelektrophorese |
| RU2180229C1 (ru) * | 2001-01-19 | 2002-03-10 | ООО Научно-производственное фармацевтическое предприятие "Полидан" | Способ получения растворов натрия нуклеоспермата |
| US6872530B2 (en) | 2002-04-24 | 2005-03-29 | Spectrumedix, Llc | Method for determining the presence of DNA variants using peptide nucleic acid probes |
| US7303879B2 (en) | 2003-07-31 | 2007-12-04 | Applera Corporation | Determination of SNP allelic frequencies using temperature gradient electrophoresis |
| WO2005049196A1 (ja) | 2003-11-21 | 2005-06-02 | Ebara Corporation | 液体を用いたマイクロチップ装置 |
| EP1760159A1 (de) * | 2005-08-31 | 2007-03-07 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Verfahren zur Elektrophorese von Nukleinsäuremolekülen |
| JP6992385B2 (ja) * | 2017-10-02 | 2022-01-13 | 株式会社島津製作所 | 電気泳動用分離媒体、電気泳動用試薬キット、及び電気泳動方法 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3023793B2 (ja) * | 1988-02-16 | 2000-03-21 | アプライド バイオシステムズ インコーポレイテッド | 毛管電気泳動方法及びその装置 |
| EP0486580B1 (de) * | 1989-08-19 | 1996-11-06 | QIAGEN GmbH | Verfahren und vorrichtung zur trennung und detektion von komponenten eines stoffgemisches durch temperaturgradienten-gelelektrophorese |
| US5066382A (en) * | 1990-01-25 | 1991-11-19 | Spectra-Physics, Inc. | Thermal control for capillary electrophoresis apparatus |
-
1994
- 1994-09-12 IT ITMI941864A patent/IT1271005B/it active IP Right Grant
-
1995
- 1995-09-11 AU AU35652/95A patent/AU689648B2/en not_active Ceased
- 1995-09-11 EP EP95932707A patent/EP0781411A1/de not_active Withdrawn
- 1995-09-11 JP JP8509892A patent/JP3015106B2/ja not_active Expired - Lifetime
- 1995-09-11 WO PCT/EP1995/003561 patent/WO1996008715A1/en not_active Application Discontinuation
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9608715A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH10502738A (ja) | 1998-03-10 |
| IT1271005B (it) | 1997-05-26 |
| ITMI941864A0 (it) | 1994-09-12 |
| ITMI941864A1 (it) | 1996-03-12 |
| AU689648B2 (en) | 1998-04-02 |
| JP3015106B2 (ja) | 2000-03-06 |
| AU3565295A (en) | 1996-03-29 |
| WO1996008715A1 (en) | 1996-03-21 |
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