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WO2002056003A2 - Procede et formulations pour la separation de macromolecules biologiques - Google Patents

Procede et formulations pour la separation de macromolecules biologiques Download PDF

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Publication number
WO2002056003A2
WO2002056003A2 PCT/US2002/001132 US0201132W WO02056003A2 WO 2002056003 A2 WO2002056003 A2 WO 2002056003A2 US 0201132 W US0201132 W US 0201132W WO 02056003 A2 WO02056003 A2 WO 02056003A2
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
matrix formulation
lpa
matrix
linear polyacrylamide
Prior art date
Application number
PCT/US2002/001132
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English (en)
Other versions
WO2002056003A3 (fr
Inventor
Richard Hammond
Sam Chen
Marie C. Ruiz-Martinez
Kendra Swirsding
Yi-Neng Wu
Jan F. Simons
Bruce Taillon
Original Assignee
Curagen 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 Curagen Corporation filed Critical Curagen Corporation
Priority to AU2002234252A priority Critical patent/AU2002234252A1/en
Publication of WO2002056003A2 publication Critical patent/WO2002056003A2/fr
Publication of WO2002056003A3 publication Critical patent/WO2002056003A3/fr

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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
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention relates generally to methods and formulations for the separation of biological macromolecules such as polynucleotides.
  • Capillary electrophoresis has demonstrated its advantages over standard slab gel based electrophoretic techniques as a rapid, high-throughput and high-resolution method for separation of biological macromolecules, such as proteins, peptides and nucleic acids
  • Capillary gel electrophoresis (CGE) is the CE-analog of traditional slab-gel electrophoresis and is most often used for size-based separation of biological macromolecules such as oligonucleo tides, DNA restriction fragments and proteins. The separation is performed by filling the capillary with a sieving matrix, for example, cross-linked polyacrylamide, agarose or linear polymer solutions.
  • the invention is based in part on the discovery of new matrix formulations for easily resolving nucleic acids 15-90 polynucleotides in length.
  • the invention features a replaceable matrix formulation consisting of 3M-8M urea, a buffer, at least one denaturant, a linear polyacrylamide (LPA) of about 2.5xl0 6 - 5.0 ⁇ l0 6 daltons (referred to herein as high MW LPA or long LPA), and a linear polyacrylamide of about 1.3xl0 5 - 2.0 10 5 daltons (referred to herein as low MW LPA or short LPA).
  • LPA linear polyacrylamide
  • the high MW LPA is provided in a size range of about 3.0 xlO 6 - 4.0 xlO 6 daltons.
  • the low MW LPA is provided in a size range of about 1.5xl0 5 - 1.8x10 s daltons, e.g., about 1.6 xlO 5 - 1.7 xlO 5 daltons.
  • the concentration of high MW LPA contained in the matrix formulation can range from about 1.0% to about 3.0% (w/w). In other embodiments, the concentration of low MW LPA contained in the matrix formulation can range from about 1.0% to about 3.0% (w/w). Each LPA concentration can be varied independently.
  • the invention includes a matrix formulation having an organic denaturant.
  • the organic denatuaraunt can be, e.g., pyrrolidinone (including 2-pyrrolidinone), N',N'-dimethylacetamide, and N',N'-dimethylformamide.
  • concentration of organic denaturant can range from 5% to 20% (w/w).
  • the invention also provides a method of detecting a nucleic acid in a population of nucleic acid molecules by providing a capillary electrophoresis device with a sieving matrix formulation that includes double-LPA matrix, an organic denaturant, and urea, and introducing a first nucleic acid into the formulation.
  • the first nucleic acid is transported through the matrix formulation and detected. Transporting is preferably performed by electrophoresis, e.g., capillary electrophoresis.
  • a second nucleic acid is introduced into the matrix formulation, transported through the matrix formulation and detected.
  • the first nucleic acid and/or the second nucleic acid include a detectable label, e.g., a radioactive or fluorescent label.
  • a "double-LPA matrix” is any sieving matrix that includes a mixture of high MW LPA and low MW LPA in addition to at least one denaturant, a buffer, and 3M-8M urea.
  • the invention includes resolving nucleic acids in a population of nucleic acid molecules that includes nucleotides from at least 15 nucleotides to at least about 75 nucleotides in length.
  • the separation results in a resolution per base higher than 1.0.
  • the method involves introducing a second population of nucleic acid molecules into the sieving matrix formulation.
  • the nucleic acids are separated using an electrophoresis device that is operated at a run voltage of between 10kV-14kV.
  • the electrophoresis device is operated at a temperature ranging between 40°C and 60°C.
  • the matrix formulations are used to determine the nucleotide sequence of at least a portion of at least one member of the population of nucleic acid molecules.
  • FIG. 1 is an electrophoretogram showing the elution profile of rox-labeled DNA ladder through a standard, high MW LPA sieving matrix.
  • FIG. 2 is an electrophoretogram showing the elution profile of rox-labeled DNA ladder through a double-LPA sieving matrix.
  • FIG. 3 is an electrophoretogram showing the elution profile of rox-labeled DNA ladder through a double-LPA sieving matrix containing pyrrolidinone.
  • FIG. 4 is a histogram comparing the resolution of DNA ladder peaks for three concentrations of low MW LPA added to the standard, high MW LPA matrix.
  • FIG. 5 is a histogram comparing the resolution of DNA ladder peaks for three concentrations of pyrrolidinone added to sieving matrix.
  • FIG. 6 is an electrophoretogram showing the elution profile of four consecutive injections of SNPs in one electrophoresis lane.
  • Formulations of the invention include two linear polyacrylamide solutions, referred to herein as high MW polyacrylamide and low MW polyacrylamide, an organic denaturant, a buffer, and urea.
  • High and low molecular weight formulations are known in the art.
  • Long LPA formulations are discussed in, e.g., Ruiz-Martinez et al, Anal. Chem. 70:1516-27, 1998; Goetzinger et al., Electrophoresis 19:242-48, 1998, and in WOOO/28314.
  • LPA can be obtained from commercial vendors such as Molecular Dynamics.
  • Low MW LPA and high MW LPA are combined in proportions designed to optimize separation of a pool of nucleic acid sequences of a desired target nucleic acid of a desired size range.
  • the target range is 15-100, or 20-90, or 30-75, nucleotides in length.
  • the high MW LPA is a molecular weight of about 2.5x10 6 -
  • the high MW LPA is provided in a size range of about 3.0 x 10 6 - 4.0 x 10 6 daltons.
  • the low MW LPA is provided in a size range of about 1.5xl0 5 - 1.8xl0 5 daltons, e.g., about 1.6 xlO 5 - 1.7 xlO 5 daltons.
  • suitable formulations include those that include low MW LPA at 1-3% w/w and high MW LPA at 1-3% w/w.
  • the formulations preferably includes an organic denaturants in order to maximize high resolution and enhanced denaturation of single stranded DNA fragments.
  • the organic denaturant can include, e.g., (i) 2-pyrrolidinone, (ii) N',N'-dimethylformamide, and (iii)
  • N',N'-dimethylacetamide at 5-40% (w/w).
  • the formulations can also include 3M-8 M urea, with a suitable buffer. Preferred concentrations of urea are 6M-7M. Buffers typically used for LPA formulations systems can be used, so long as they allow for resolution of molecules in a desired size range.
  • An example of a suitable buffer 0.1-300 mM TAPS-Tris-EDTA.
  • the formulations described herein can be used in any application for which resolution of small nucleic acid fragments is desired.
  • the formulations can be used in, e.g., sizing a nucleic acid of interest, fingerprinting (including dideoxyfingerprinting), identification of short tandem repeats (STRs), detection of single nucleotide polymorphisms (SNPs), detection of cDNA copies of RNA molecules (include applications for detecting levels of one or more RNA sequences in a population of interest).
  • the formulations are used to resolve nucleic acids that have been analyzed with single base extension (SBE) chemistry with fluorescent labeled di- deoxynucleotides.
  • SBE single base extension
  • Suitable primer lengths to use for single base extension is 20 to 40 nucleotides.
  • SBE chemistry a nucleotide fragment (-200 bases) near the polymorphic region of the DNA is amplified using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Shrimp alkaline phosphatase and E. coli exonuclease I are added to the reaction mixture to digest the remaining dNTP's and PCR primers. After digestion, the sample is heated to 95 °C to destroy the remaining enzymes and the single base extension reaction is performed using fluorescent labeled di-deoxynucleotides.
  • allele-specific ddNTPs are dye-labeled, a different dye for each allele.
  • a primer is added that will anneal adjacent to the single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • unincorporated ddNTPs are removed (e.g., using chromatographic separation such as SEPHADEXTM filtration).
  • Figure 1 shows the electrophoresis run of rox-labeled DNA ladder, containing fragments of 15bp, 16bp, 17bp, 20bp, 21bp, 25bp, 32bp, 33bp, 41bp, 42bp, and 43bp.
  • the sieving matrix formulation for this electrophoresis run was a standard, linear polyacrylamide ("LPA") obtained from Molecular Dynamics (also referred to herein as high MW LPA or long LPA). Individual peaks corresponding to the various fragments could not be resolved using these formulations.
  • LPA linear polyacrylamide
  • Double-LPA matrixes Nine different matrix formulations that included two types of linear polyacrylamide (“double-LPA matrixes”) were prepared to evaluate the separation of short nucleotide fragments.
  • Each matrix formulation contained 2% of a first linear polyacrylamide solution with an average molecular weight range of 2.5x10° to 5.0x10° daltons and 7M Urea.
  • the formulations additionally included varying amounts of a second linear polyacrylamide solution with an average molecular weight of about l. ⁇ xlO 5 daltons (also referred to herein as low molecule weight or short LPA), and varying amounts of the denaturant pyrrolidinone were added as shown in Table 1.
  • FIG. 2 shows the electrophoresis run of rox-labeled DNA ladder, containing fragments of 15bp, 16bp, 17bp, 20bp, 21bp, 25bp, 32bp, 33bp, 41bp, 42bp, and 43bp.
  • the sieving matrix formulation was 2% high MW LPA, 1.5% low MW LPA, and 7M urea. No pyrrolidinone was present.
  • FIG. 3 shows the electrophoresis run of rox-labeled DNA ladder, containing fragments of 15bp, 16bp, 17bp, 20bp, 21bp, 25bp, 32bp, 33bp, 41bp, 42bp, and 43bp.
  • the sieving matrix formulation for this electrophoresis separation included 2% high MW LPA, 2.5% low MW LPA, 7M urea and 5% pyrrolidinone.
  • the addition of pyrrolidinone retarded the migration of the nucleotide fragments.
  • Example 3 Effect of varying the amount of low MW LPA in a double-LPA matrix
  • FIG. 4 presents shows a comparison of resolution for selected DNA ladder peaks for different concentrations of low MW LPA added to the standard, high MW LPA matrix.
  • Matrix 2 1.5% added low MW LPA
  • matrix 5 2.0% added low MW LPA
  • matrix 8 2.5%o added low MW LPA. Addition of low MW LPA to 2.5% provides even resolution across the DNA size range from 21bp to 41bp.
  • Example 4 Effect of varying the amount of pyrrolidinone in a double-LPA matrix
  • FIG. 5 shows a comparison of resolution for selected DNA ladder peaks for different concentrations of pyrrolidinone added to sieving matrix.
  • Each nucleic acid was separated in a matrix that included 5% added pyrrolidinone (Matrix 7), 10%> added pyrrolidinone (Matrix 8), or 15%) added pyrrolidinone (Matrix 9).
  • the results for matrix tested are presented as Matrix 7-Matrix 8-Matrix-9 (left to right).
  • the double-LPA matrix formulation provides single base resolution down to 15bp. This allows for a large increase in genotyping throughput by multiplexing several SNPs together, each of different primer length. Multiplexing can also be accomplished through multiple injections during a single electrophoresis run.
  • the double-LPA sieving matrix provides improved resolution of short nucleotide fragments coupled with short run times, allowing for a greater number of injections to be multiplexed.
  • FIG. 6 An application of multiplexing using double-LPA formulations is illustrated in FIG. 6. This figure presents the results of introducing four separate nucleic acids into a single matrix. No loss of resolution was observed. Additional peaks observed in the figure are due to unincorporated ddNPTs.
  • FIG. 6 further shows that multiple injections can be performed in one lane of electrophoresis.
  • the inserted table in Figure 6 illustrates that for the separation of these small DNA fragments the resolution is preserved even with the use of multiple injections.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion 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)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des procédés et des formulations pour la séparation de macromolécules biologiques, telles que des polynucléotides.
PCT/US2002/001132 2001-01-12 2002-01-14 Procede et formulations pour la separation de macromolecules biologiques WO2002056003A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002234252A AU2002234252A1 (en) 2001-01-12 2002-01-14 Method and formulations for the separation of biological macromolecules

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US26148901P 2001-01-12 2001-01-12
US60/261,489 2001-01-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004020991A1 (fr) * 2002-09-02 2004-03-11 Proteome Systems Intellectual Property Pty Ltd Gel d'electrophorese presentant des proprietes de gonflement ameliorees
EP2362212A3 (fr) * 2009-03-18 2012-03-28 Agilent Technologies, Inc. PDMA et cellulose dérivée en tant que support de séparation pour ADN

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6554985B2 (en) * 1998-11-10 2003-04-29 Curagen Corporation Methods and formulations for the separation of biological macromolecules

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004020991A1 (fr) * 2002-09-02 2004-03-11 Proteome Systems Intellectual Property Pty Ltd Gel d'electrophorese presentant des proprietes de gonflement ameliorees
EP2362212A3 (fr) * 2009-03-18 2012-03-28 Agilent Technologies, Inc. PDMA et cellulose dérivée en tant que support de séparation pour ADN

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WO2002056003A3 (fr) 2003-05-15
AU2002234252A1 (en) 2002-07-24

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