WO2002056003A2

WO2002056003A2 - Method and formulations for the separation of biological macromolecules

Info

Publication number: WO2002056003A2
Application number: PCT/US2002/001132
Authority: WO
Inventors: Richard Hammond; Sam Chen; Marie C. Ruiz-Martinez; Kendra Swirsding; Yi-Neng Wu; Jan F. Simons; Bruce Taillon
Original assignee: CuraGen Corp
Current assignee: CuraGen Corp
Priority date: 2001-01-12
Filing date: 2002-01-14
Publication date: 2002-07-18
Anticipated expiration: 2003-07-12
Also published as: WO2002056003A3; AU2002234252A1

Abstract

Provided are methods and formulations for the separation of biological macromolecules such as polynucleotides.

Description

METHOD AND FORMULATIONS FOR THE SEPARATION OF BIOLOGICAL

MACROMOLECULES

FIELD OF THE INVENTION

The present invention relates generally to methods and formulations for the separation of biological macromolecules such as polynucleotides.

BACKGROUND OF THE INVENTION

Capillary electrophoresis (CE) 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.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery of new matrix formulations for easily resolving nucleic acids 15-90 polynucleotides in length.

Accordingly, in one aspect, the invention features a replaceable matrix formulation consisting of 3M-8M urea, a buffer, at least one denaturant, a linear polyacrylamide (LPA) of about 2.5xl0⁶ - 5.0χl0⁶ daltons (referred to herein as high MW LPA or long LPA), and a linear polyacrylamide of about 1.3xl0⁵ - 2.0 10⁵ daltons (referred to herein as low MW LPA or short LPA). In some embodiments, the high MW LPA is provided in a size range of about 3.0 xlO⁶ - 4.0 xlO⁶ daltons. In some embodiments, the low MW LPA is provided in a size range of about 1.5xl0⁵ - 1.8x10^s daltons, e.g., about 1.6 xlO⁵ - 1.7 xlO⁵ daltons.

In some embodiments, 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.

In various embodiments, 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. The 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.

In some embodiments, a second nucleic acid is introduced into the matrix formulation, transported through the matrix formulation and detected.

Preferably, the first nucleic acid and/or the second nucleic acid include a detectable label, e.g., a radioactive or fluorescent label.

As used herein, 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.

In one embodiment, 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. In another embodiment, the separation results in a resolution per base higher than 1.0. In a further embodiment, the method involves introducing a second population of nucleic acid molecules into the sieving matrix formulation.

In another embodiment, the nucleic acids are separated using an electrophoresis device that is operated at a run voltage of between 10kV-14kV. In still further embodiment, the electrophoresis device is operated at a temperature ranging between 40°C and 60°C. In one embodiment, 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION

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. In some embodiments, the target range is 15-100, or 20-90, or 30-75, nucleotides in length.

In various embodiments, the high MW LPA is a molecular weight of about 2.5x10⁶ -

5.0x10⁶ daltons (referred to herein as high MW LPA or long LPA). For example, in some embodiments, the high MW LPA is provided in a size range of about 3.0 x 10⁶ - 4.0 x 10⁶ daltons.

In some embodiments, the low MW LPA is provided in a size range of about 1.5xl0⁵ - 1.8xl0⁵ daltons, e.g., about 1.6 xlO⁵ - 1.7 xlO⁵ daltons.

Examples of 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. Thus, 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).

In one embodiment, the formulations are used to resolve nucleic acids that have been analyzed with single base extension (SBE) chemistry with fluorescent labeled di- deoxynucleotides. Suitable primer lengths to use for single base extension is 20 to 40 nucleotides. In SBE chemistry a nucleotide fragment (-200 bases) near the polymorphic region of the DNA is amplified using polymerase chain reaction (PCR). 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.

These allele-specific ddNTPs are dye-labeled, a different dye for each allele. To this reaction a primer is added that will anneal adjacent to the single nucleotide polymorphism (SNP). After primer extension, unincorporated ddNTPs are removed (e.g., using chromatographic separation such as SEPHADEX™ filtration).

Among the advantages of the invention is that multiple samples can be processed through a single formulation. This multiplexing allows for accurate, rapid and economical analysis of multiple samples.

The formulations and methods described herein can be used in art-recognized apparatuses for performing high-throughput, DNA sequencer that using capillary electrophoresis. Apparatuses for performing LPA separation are described in, e.g., Ruiz-Martinez et al., Anal. Chem. 70:1516-27, 1998; Goetzinger et al., Electrophoresis 19:242-48, 1998, WOOO/28314, and can be obtained from commercial vendors such as Molecular Dynamics.

The invention will be further illustrated in the following non-limiting examples.

EXAMPLES Example 1. Separation of short nucleotide fragments using high molecular weight linear polyacrylamide

The ability of various matrix formulations to resolve nucleic acids of various molecular weights was examined. 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.

Example 2. Preparation of matrix formulations including two linear polyacrylamide solutions

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.δxlO⁵ 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.

Table 1. Matrix Formulations Containing Long and Short LPA

Example 3. Resolution of nucleic acid fragments using matrix formulations containing long and short LPA

The ability of matrix formulations containing both long and short LPA to resolve short nucleic acid fragments was examined.

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.

The addition of low MW LPA to the standard, high MW LPA matrix improved the resolution of short nucleotide fragments relative to the resolution seen with high MW LPA alone (see FIG. 1). Broad peaks were detected for fragments of 15bp-20bp.

Example 4. Effect of adding pyrrolidinone to a double-LPA matrix

The ability of matrix formulations containing varying amounts of pyrrolidinone to resolve short nucleic acid fragments was examined. 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. DNA fragments of 15bp-20bp eluted later than the leading electrolyte zone, and the broad peaks seen in FIG. 2 were not observed. All of the rox-labeled DNA ladder fragments ranging from 15bp-34bp were resolved.

Example 3. Effect of varying the amount of low MW LPA in a double-LPA matrix

The ability of matrix formulations containing varying amounts of short LPA to resolve short nucleic acid fragments was examined.

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, and 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

The ability of matrix formulations containing varying amounts of pyrrolidinone to resolve short nucleic acid fragments was examined.

FIG. 5 shows a comparison of resolution for selected DNA ladder peaks for different concentrations of pyrrolidinone added to sieving matrix. Resolution of nucleic acids 21, 25, 32, 33, and 41 nucleotides in length. 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). For each nucleic acid tested, the results for matrix tested are presented as Matrix 7-Matrix 8-Matrix-9 (left to right).

The greatest resolution was observed using 5% pyrrolidinone, indicating that DNA fragments greater than 20bp are sufficiently removed from interfering electrolyte zones.

This minimum pyrrolidinone concentration also allowed for shorter run times.

Example 5. Multiplexing using double-LPA formulations

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.

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.

OTHER EMBODIMENTS

From the above description, the essential characteristics of the present invention can be ascertained. Without departing from the spirit and scope thereof, various changes and modifications of the invention can be made to adapt it to various usages and conditions. For example, the formulations and methods disclosed herein can also be used to resolve other molecules such as polypeptides. Additional embodiments are also within the claims.

Claims

What is claimed is:

1. A replaceable matrix formulation comprising: a first linear polyacrylamide of about 2.5 xlO⁶ - 5.0^χl0⁶D; a second linear polyacrylamide of about 1.5χl0⁵ - 2.0x10⁵ D; at least one denaturant; a buffer; and 3M-8M urea.

2. The matrix formulation of claim 1, wherein the first linear polyacrylamide concentration is from 1.0% to about 3.0%> (w/w).

3. The matrix formulation of claim 1, wherein the second linear polyacrylamide concentration is from about 1.0% to about 3.0%.

4. The matrix formulation of claim 2, wherein the second linear polyacrylamide concentration is from about 1.0% to about 3.0%>.

5. The matrix formulation of claim 1, wherein the denaturant is organic.

6. The matrix formulation of claim 5, wherein the organic denaturant is selected from the group consisting of 2-pyrrolidinone, N',N'-dimethylacetamide, and N',N'-dimethylformamide.

7. The matrix formulation of claim 4, wherein the organic denaturant concentration ranges between 5%-20% (w/w).

8. The matrix formulation of claim 5, wherein the organic denaturant is

2-pyrrolidinone.

9. A method of detecting a nucleic acid in a population of nucleic acid molecules, the method comprising providing a capillary electrophoresis device comprising a matrix formulation that includes a first linear polyacrylamide of about 2.5χl0⁶ - 5.0^χl0⁶D, a second linear polyacrylamide of about 1.5xl0⁵ - 2.0xl0⁵D, at least one denaturant, a buffer, and 3M-

8M urea; introducing the nucleic acid population into said matrix formulation; and resolving the nucleic acids in said population by transporting said population through said matrix formulation; and detecting a resolved nucleic acid.

10. The method of claim 9, wherein said first population of nucleic acid molecules includes nucleic acids ranging in size from about 15 nucleotides to about 100 nucleotides in length.

11. The method of claim 9, wherein the method resolves nucleic acids whose lengths differ by 1 nucleotide.

12. The method of claim 9, wherein said first nucleic acid has a detectable label.

13. The method of claim 9, wherein said method further comprises introducing a second nucleic acid into said matrix formulation.

14. The method of claim 9, wherein the first linear polyacrylamide concentration is from about 1.0% to about 3.0%.

15. The method of claim 9, wherein the second linear polyacrylamide concentration is from about 1.0% to about 3.0%.

16. The method of claim 9, wherein said method further involves determining the nucleotide sequence of at least a portion of at least one member of said population of nucleic acid molecules.

17. A method of resolving nucleic acids in a population of nucleic acid molecules, the method comprising providing a capillary electrophoresis device comprising a matrix formulation that includes a first linear polyacrylamide, a second linear polyacrylamide, at least one denaturant, a buffer, and 3M-8M urea; introducing a first nucleic acid into said matrix formulation; transporting said first nucleic acid through said matrix formulation; introducing a second nucleic acid into said matrix formulation; and transporting said nucleic acid through said matrix formulation; and detecting the first and second nucleic acids.

18. The method of claim 17, wherein the first and second nucleic acids are from at least about 15 nucleotides to at least about 75 nucleotides in length.

19. The method of claim 17, wherein the first nucleic acid includes a detectable label.

20. The method of claim 17, wherein the second nucleic acid includes a detectable label.