EP0781411A1 - Separation d'acides nucleiques dans des solutions polymeres visqueuses au moyen d'une electrophorese capillaire a gradients thermiques - Google Patents

Separation d'acides nucleiques dans des solutions polymeres visqueuses au moyen d'une electrophorese capillaire a gradients thermiques

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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Withdrawn
Application number
EP95932707A
Other languages
German (de)
English (en)
Inventor
Pier Giorgio Righetti
Cecilia Gelfi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0781411A1 publication Critical patent/EP0781411A1/fr
Withdrawn legal-status Critical Current

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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)
  • Biochemistry (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (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)

Abstract

On décrit l'utilisation de gradients thermiques variables dans le temps, destinés à la séparation de fragments d'ADN amplifiés à l'aide d'une amplification en chaîne par polymérase (PCR) et à la fois normaux et contenant des mutations ponctuelles, au moyen d'une électrophorèse de zone sur colonne capillaire en présence de solutions polymères visqueuses. Dans ce système, on obtient la régulation des températures dans les zones capillaires à l'aide d'un logiciel approprié et les fragments sont révélés soit par leur pouvoir absorbant naturel du rayonnement ultraviolet à 254 nm, soit par une fluorescence induite par laser. Il est également possible de travailler avec des solutions visqueuses de polyacrylamides, notamment de polyacrylamides contenant des monomères substitués par N, tels que N-méthylacrylamide et N-acryloyle amino éthoxy éthanol. On décrit des procédés de production de polyacrylamides à courte chaîne et à faible viscosité que l'on peut remplacer après chaque opération.
EP95932707A 1994-09-12 1995-09-11 Separation d'acides nucleiques dans des solutions polymeres visqueuses au moyen d'une electrophorese capillaire a gradients thermiques Withdrawn EP0781411A1 (fr)

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 (fr) 1994-09-12 1995-09-11 Separation d'acides nucleiques dans des solutions polymeres visqueuses au moyen d'une electrophorese capillaire a gradients thermiques

Publications (1)

Publication Number Publication Date
EP0781411A1 true EP0781411A1 (fr) 1997-07-02

Family

ID=11369544

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95932707A Withdrawn EP0781411A1 (fr) 1994-09-12 1995-09-11 Separation d'acides nucleiques dans des solutions polymeres visqueuses au moyen d'une electrophorese capillaire a gradients thermiques

Country Status (5)

Country Link
EP (1) EP0781411A1 (fr)
JP (1) JP3015106B2 (fr)
AU (1) AU689648B2 (fr)
IT (1) IT1271005B (fr)
WO (1) WO1996008715A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
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
CA2407124C (fr) * 2000-04-25 2010-09-28 Spectrumedix Corporation Electrophorese de molecules biologiques effectuee sans denaturants dans des conditions a haute temperature
EP1320745A4 (fr) * 2000-09-01 2008-10-01 Applera Corp Systeme et procede d'electrophorese capillaire a gradient de temperature
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
TW200536601A (en) 2003-11-21 2005-11-16 Ebara Corp Micorfluidic treatment method and device
EP1760159A1 (fr) * 2005-08-31 2007-03-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé ameliore pour electrophorese des acides nucleiques
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)

* Cited by examiner, † Cited by third party
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
ATE145010T1 (de) * 1989-08-19 1996-11-15 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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9608715A1 *

Also Published As

Publication number Publication date
ITMI941864A1 (it) 1996-03-12
AU689648B2 (en) 1998-04-02
JPH10502738A (ja) 1998-03-10
ITMI941864A0 (it) 1994-09-12
AU3565295A (en) 1996-03-29
IT1271005B (it) 1997-05-26
WO1996008715A1 (fr) 1996-03-21
JP3015106B2 (ja) 2000-03-06

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