WO1994023092A1 - Gel de sequencage ameliore - Google Patents

Gel de sequencage ameliore Download PDF

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Publication number
WO1994023092A1
WO1994023092A1 PCT/US1994/003274 US9403274W WO9423092A1 WO 1994023092 A1 WO1994023092 A1 WO 1994023092A1 US 9403274 W US9403274 W US 9403274W WO 9423092 A1 WO9423092 A1 WO 9423092A1
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WO
WIPO (PCT)
Prior art keywords
gel
ethylene glycol
formamide
gels
sequencing gel
Prior art date
Application number
PCT/US1994/003274
Other languages
English (en)
Inventor
Carl W. Fuller
Original Assignee
United States Biochemical Corporation
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 United States Biochemical Corporation filed Critical United States Biochemical Corporation
Priority to AU65250/94A priority Critical patent/AU6525094A/en
Publication of WO1994023092A1 publication Critical patent/WO1994023092A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/52—Amides or imides
    • C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F20/56—Acrylamide; Methacrylamide
    • 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
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • This invention relates to gels suitable for separating nucleic acid fragments differing by only one base over a range of fragments containing between 10 and 1,000 bases.
  • Gels which are formed from polyacryla ide, or derivatives thereof, are commonly used for separating DNA fragments (or bands) having a size between 10 and 1,000 bases which differ by only one base. These gels are commonly formed with formamide, or a mixture of formamide and urea, to ensure that the DNA fragments running within the gel are in a single-stranded or denatured form. Examples of such gels are described by Sugihara et al., U.S. Patent 4,844,786, which also describes use of gels containing between 5 to 40 wt/v % of a polyol, such as glycerol or ethylene glycol, to prevent prolongation and distortion of protein bands.
  • a polyol such as glycerol or ethylene glycol
  • This invention relates to novel sequencing gels in which ethylene glycol is used in place of urea, or at least a portion of the formamide normally used as a denaturant in the gel.
  • the invention thus concerns use of a mixture of ethylene glycol and formamide to achieve substantially the same result as ethylene glycol alone, without the expense of using only formamide (such mixtures range from mixtures of 50%/50% (v/v) to 100%/0% ethylene glycol/formamide, preferably with about 70% to 80% ethylene glycol) . It is important that such gels not contain urea.
  • the ethylene glycol lowers the melting temperature of DNA within the gel sufficiently to allow its use as a denaturant for electrophoresis of single-stranded DNA fragments.
  • the melting point of most DNAs in 70% ethylene glycol is only about 45°C, and those DNAs in which deoxyguanine is replaced with deazaguanine or deoxyinosine, or which contain ⁇ -thio nucleotides in place of standard oxygen-containing nucleotides, may have lower melting points.
  • Such gels can be run using the diol-tolerant buffers described by Fuller, U.S. Serial No. 07/928,852, filed August 10, 1992, hereby incorporated by reference herein.
  • the invention features a DNA sequencing gel containing ethylene glycol and formamide in an total amount between 50% and 90% (v/v) , with the formamide forming an equal or lesser amount than the ethylene glycol.
  • the gel includes polyacrylamide (or other polymerized monomers other than acrylamide) and buffering components commonly used in polyacrylamide DNA seqencing gels.
  • the invention features a method for separating nucleic acid fragments in a gel by providing a gel containing at least 50%-90% (v/v) ethylene glycol and/or formamide, as described above.
  • the method includes heating the gel to a temperature of at least 40°-60°C.
  • Applicant has determined that other non-ionic compounds which are soluble in water at a concentration of 50% w/v or v/v, and which do not react with other gel components, such as acrylamide, tris or taurine, are also useful in gel formation.
  • these include not only formamide and dimethyl formamide, but also dimethyl sulfoxide, N-methyl pyrrolidone, hexamethylphosphoramide, acetamide, propylene glycol, glycerol, 1,3-propanediol, tetramethylurea, and polyvinylalcohol.
  • these components can also be used in conjunction with the ethylene glycol.
  • the invention features a kit for formation of an electrophoretic gel containing, in a separate container, ethylene glycol *
  • the kit includes acrylamide monomers, and catalysts useful for forming the gel, e.g. , ammonium persulfate and N,N,N',N'- tetramethylethylenediamine, and a gel buffer, such as that described by Fuller, supra .
  • Applicant provides a novel gel useful for separation of nucleic acid fragments.
  • the gel is formed with ethylene glycol or its equivalent, and does not suffer from the decomposition or isomerization problems that may occur in a urea-based gel (which applicant believes isomerizes to form ammonium cyanate) .
  • the ethylene glycol is relatively inexpensive.
  • the ethylene glycol is also less viscous than formamide and thus, more readily used either alone or in conjunction with formamide, or its equivalent, in formation of a polyacrylamide gel. Its use in a gel allows formation of pre-cast gels which will not lose their characteristic ability to separate nucleic acid fragments, and can be provided in a pre-mixed gel solution.
  • a stable gel-forming solution can be prepared several weeks ahead of the time for its use, the gel so formed is stable for several weeks, the cost of the gel is less than a urea or formamide gel, and the gel may have improved denaturation characteristics over urea, and thus aid resolution of compressions in sequencing mixtures.
  • the invention features solutions which can be used to form a sequencing gel.
  • solutions contain the compounds noted above with either acrylamide monomers or other monomers.
  • Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
  • Description of the Preferred Embodiments The following is a non-limiting example of a gel of this invention. Those in the art will recognize it as but one example of many within the spirit of the invention described above.
  • Example Buffer Glycerol tolerant gel buffer ("20-fold concentrate”) : 216 g tris base, 72 g taurine, 4 g Na 2 EDTA «2H 2 0, H 2 0 to 1000 ml.
  • Gelling solution 7.6 g acrylamide, 0.4 g N,N'- methylenebisacrylamide, 60 ml ethylene glycol, 3.5 ml buffer concentrate (above) , water to 100 ml total volume.
  • For polymerization add 1 ml of 10% ammonium persulfate, 0.08 ml N,N,N' ,N'-tetramethylethylenediamine.
  • Cast gel 0.4 mm thick, 35 cm wide, 43 cm long; gel polymerizes in about 10 minutes.
  • Electrophoresis Running buffer is 35 ml of buffer concentrate (above) diluted to 1 liter. Gel is run at 60 watts constant power, final voltage approximately 3000 volts, 6.5 hours.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)

Abstract

L'invention concerne un procédé de formation d'un gel de séquençage d'ADN par formation d'un gel contenant de l'éthylène glycol et du formamide en une dose comprise entre 50 % et 90 %.
PCT/US1994/003274 1993-03-26 1994-03-25 Gel de sequencage ameliore WO1994023092A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU65250/94A AU6525094A (en) 1993-03-26 1994-03-25 Improved sequencing gel

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3760893A 1993-03-26 1993-03-26
US08/037,608 1993-03-26

Publications (1)

Publication Number Publication Date
WO1994023092A1 true WO1994023092A1 (fr) 1994-10-13

Family

ID=21895264

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/003274 WO1994023092A1 (fr) 1993-03-26 1994-03-25 Gel de sequencage ameliore

Country Status (2)

Country Link
AU (1) AU6525094A (fr)
WO (1) WO1994023092A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922185A (en) * 1994-03-31 1999-07-13 Novel Experimental Technology, Inc. System for pH-neutral stable electrophoresis gel
US6143154A (en) * 1994-03-31 2000-11-07 Novex System for PH-neutral stable electrophoresis gel
WO2001002602A2 (fr) * 1999-07-05 2001-01-11 Lion Bioscience Ag Nucleotides de sequençage cyclique de matrices riches en gc
CN1063797C (zh) * 1996-07-24 2001-03-28 郭大玮 分析人类基因组短串联重复顺序多态性的电泳系统
WO2004024958A1 (fr) * 2002-09-16 2004-03-25 Corning Incorporated Compositions d'encre d'acide nucleique a disposer sur un support solide
US6783651B1 (en) 1994-03-31 2004-08-31 Invitrogen Corporation System for pH-neutral stable electrophoresis gel
WO2009093054A1 (fr) * 2008-01-24 2009-07-30 Lab901 Limited Composition de gel
US8591713B2 (en) 2008-06-06 2013-11-26 Agilent Technologies, Inc. Electrophoresis cassette

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0137753A1 (fr) * 1983-09-14 1985-04-17 Fuji Photo Film Co., Ltd. Milieu pour électrophorèse
EP0139471A2 (fr) * 1983-09-14 1985-05-02 Fuji Photo Film Co., Ltd. Médium pour l'électrophorèse
US4548869A (en) * 1983-03-11 1985-10-22 Fuji Photo Film Co., Ltd. Element for electrophoresis
EP0165069A2 (fr) * 1984-06-14 1985-12-18 Fuji Photo Film Co., Ltd. Milieu pour l'électrophorèse
US4699705A (en) * 1983-05-19 1987-10-13 Director Of The Finance Division Minister's Secretariat Science And Technology Agency Element for electrophoresis using aqueous polyacrylamide gel
US4722777A (en) * 1984-06-30 1988-02-02 Fuji Photo Film Co., Ltd. Improvement of electrophoretic element using polyacrylamide gel
US4834854A (en) * 1987-08-20 1989-05-30 Fuji Photo Film Co., Ltd. Method of making an electrophoresis medium
US4844786A (en) * 1987-02-26 1989-07-04 Fuji Photo Film Co., Ltd. Means for electrophoresis
US5190629A (en) * 1987-07-16 1993-03-02 Fuji Photo Film Co., Ltd. Electrophoresis medium membrane

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4548869A (en) * 1983-03-11 1985-10-22 Fuji Photo Film Co., Ltd. Element for electrophoresis
US4699705A (en) * 1983-05-19 1987-10-13 Director Of The Finance Division Minister's Secretariat Science And Technology Agency Element for electrophoresis using aqueous polyacrylamide gel
EP0137753A1 (fr) * 1983-09-14 1985-04-17 Fuji Photo Film Co., Ltd. Milieu pour électrophorèse
EP0139471A2 (fr) * 1983-09-14 1985-05-02 Fuji Photo Film Co., Ltd. Médium pour l'électrophorèse
US4657656A (en) * 1983-09-14 1987-04-14 Fuji Photo Film Co., Ltd. Polyacrylamide medium for electrophoresis having improved elasticity
EP0165069A2 (fr) * 1984-06-14 1985-12-18 Fuji Photo Film Co., Ltd. Milieu pour l'électrophorèse
US4722777A (en) * 1984-06-30 1988-02-02 Fuji Photo Film Co., Ltd. Improvement of electrophoretic element using polyacrylamide gel
US4844786A (en) * 1987-02-26 1989-07-04 Fuji Photo Film Co., Ltd. Means for electrophoresis
US5190629A (en) * 1987-07-16 1993-03-02 Fuji Photo Film Co., Ltd. Electrophoresis medium membrane
US4834854A (en) * 1987-08-20 1989-05-30 Fuji Photo Film Co., Ltd. Method of making an electrophoresis medium

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6783651B1 (en) 1994-03-31 2004-08-31 Invitrogen Corporation System for pH-neutral stable electrophoresis gel
US6059948A (en) * 1994-03-31 2000-05-09 Novex System for pH-neutral stable electrophoresis gel
US6096182A (en) * 1994-03-31 2000-08-01 Novex System for pH-neutral stable electrophoresis gel
US6143154A (en) * 1994-03-31 2000-11-07 Novex System for PH-neutral stable electrophoresis gel
US6162338A (en) * 1994-03-31 2000-12-19 Novex System for pH-neutral stable electrophoresis gel
US7967966B2 (en) 1994-03-31 2011-06-28 Life Technologies Corporation System for pH-neutral stable electrophoresis gel
US7892409B2 (en) 1994-03-31 2011-02-22 Life Technologies Corporation System for pH-neutral stable electrophoresis gel
US5922185A (en) * 1994-03-31 1999-07-13 Novel Experimental Technology, Inc. System for pH-neutral stable electrophoresis gel
US7422670B2 (en) 1994-03-31 2008-09-09 Timothy V Updyke System for pH-neutral stable electrophoresis gel
CN1063797C (zh) * 1996-07-24 2001-03-28 郭大玮 分析人类基因组短串联重复顺序多态性的电泳系统
WO2001002602A3 (fr) * 1999-07-05 2001-07-05 Lion Bioscience Ag Nucleotides de sequençage cyclique de matrices riches en gc
EP1074633A1 (fr) * 1999-07-05 2001-02-07 LION Bioscience AG Nucléotides et leur utilisation pour le séquencage de matrices riche en GC
WO2001002602A2 (fr) * 1999-07-05 2001-01-11 Lion Bioscience Ag Nucleotides de sequençage cyclique de matrices riches en gc
WO2004024958A1 (fr) * 2002-09-16 2004-03-25 Corning Incorporated Compositions d'encre d'acide nucleique a disposer sur un support solide
WO2009093054A1 (fr) * 2008-01-24 2009-07-30 Lab901 Limited Composition de gel
US8591713B2 (en) 2008-06-06 2013-11-26 Agilent Technologies, Inc. Electrophoresis cassette

Also Published As

Publication number Publication date
AU6525094A (en) 1994-10-24

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