JP2002537778A - Method for purifying DNA using immobilized capture probe - Google Patents

Abstract

(57) Abstract: A method for using a purification apparatus containing an electrophoresis medium containing an immobilized capture probe for DNA purification is disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Nucleic acid sequence information plays an important role in both basic and applied biomedical research. The nucleotide sequence of a particular portion of DNA may be beneficial with respect to the molecular mechanism of a particular disease, such as Huntington's disease. Once it is determined that a portion of the genome may be responsible for a particular disorder, elucidating the nucleotide sequence becomes crucial. Once the sequence is known, it can play a role in the therapeutic regimes provided, such as in the case of gene therapy. This is most pronounced when the cause of the disease is a gene mutation of a normal gene. One of the methodologies used to treat diseases caused by genetic mutations is the introduction of a wild-type nucleotide sequence. However, first, it must be confirmed that the gene or the abnormal form of the gene actually causes the specific disease or syndrome. This information is most often obtained by isolation and characterization of putative abnormal genes. Characterization often involves sequence analysis of the nucleotide sequence itself that defines the gene of interest. This often requires an understanding of both the wild-type or physiologically normal and mutant genes.

[0002] In practice, the quality of sequence analysis reflects, in part, the quality of the starting materials. Preparations subjected to sequence analysis are of high quality, i.e., relatively pure, and contain contaminants such as small molecules such as proteins and salts that may interfere with obtaining high quality results by sequence analysis. It is important that they are not. Current protocols use ethanol precipitation prior to sequence analysis, for example, to remove unincorporated nucleotides and salts from extension products. For extension products, it is often desirable to remove any template DNA and excess primer from the preparation prior to sequence analysis. Precipitation takes time and care must be taken to obtain a constant product yield. Nevertheless, for successful sequence analysis of the target nucleotide sequence, it is important that the target sequence preparation be as free of contaminating molecules as possible. Further, it would be advantageous to have a purification system that could select the products of a complex sequencing reaction.

SUMMARY OF THE INVENTION [0003] The present invention relates to a method for purifying a target molecule contained in a test sample. Normal,
The target molecule in the test sample is a nucleic acid molecule, in particular, for example, the Sanger method, Sanger et al.
Natl. Acad. Sci. USA, 74: 5463-5467 (1977) or cycle sequencing, Carothers, Biotechniques, 7: 494-4.
These are the products of single-stranded DNA primer extension sequencing reactions of dideoxy sequencing reactions such as 99 (1989), the disclosures of which are hereby incorporated by reference in their entirety. Once purified, these purified nucleic acid molecules can be used in a variety of ways, including subjecting them to capillary or slab gel electrophoresis for DNA sequence analysis. The nucleic acid molecule-capturing probe modified with a 5′-acrylamide group is copolymerized in an electrophoresis gel such as a polyacrylamide gel. A single-stranded target nucleic acid molecule can bind to a complementary sequence contained within a capture probe if there is sufficient complementarity between the two molecules. A double-stranded target nucleic acid molecule can bind to a complementary sequence to form a triple-stranded structure.

[0004] One or more target molecules present in a test sample can be subjected to electrophoresis in an electrophoresis gel containing immobilized capture probes. The target molecule is
Run through the gel medium until it contacts the complementary immobilized capture probe. Once the target and capture probe have contacted each other, they can hybridize to form a complex. Molecules other than the target contained in the test sample can continue to be electrophoresed, and are efficiently removed from the target molecule.

[0005] In one aspect of the invention, the target molecule is a DNA extension product formed during a primer extension sequencing reaction. The reaction mixture resulting from the primer extension sequencing reaction is loaded into a purification device, such as a microtiter plate containing multiple wells (eg, having 6, 12, 96, or 384 wells). The purification device comprises an electrophoresis gel containing immobilized capture probes complementary to at least one nucleotide sequence region contained within the target molecule. It is preferable to apply an electric field such that all negatively charged molecules move in the electrophoresis gel toward the positively charged electrode. The positively charged electrode can be contained in a positively charged electrode buffer chamber. This chamber can be used to collect molecules exiting the electrophoretic medium as a result of electrophoretic migration. The target molecule is captured by a complementary immobilized capture probe in the gel. Non-target molecules contained in the test sample pass through the gel and enter a positively charged electrode buffer (sometimes referred to herein as a collection chamber). The collection chamber can then be replaced with a new positively charged electrode buffer. A sufficient voltage can be applied to denature the hybridization complex formed by the target molecule and the capture probe to release the target molecule. The electric field can be applied using the same polarity as originally applied, which allows the free target molecule to continue to move into the collection chamber containing the new positively charged electrode buffer. Alternatively, the electric field can be reversed and the free target molecules can be drawn back into the test sample well of the purification device. The purified target molecule can be evaluated at this point and subjected to further analysis such as capillary or slab gel electrophoresis for sequence analysis.

[0006] Another aspect of the invention is a kit for purifying a primer extension sequencing reaction. The kit includes an electrophoresis medium containing one capture probe or a set of capture probes. At least one capture probe has a complementary sequence substantially at least 5 nucleotides in length to a portion of the at least one primer extension sequencing reaction product.

[0007] In yet another aspect, the kit includes a plurality of electrophoretic media for purifying a plurality of primer extension sequencing reactions. Each electrophoretic medium in the kit contains one capture probe or set of capture probes.
At least one capture probe in each medium has a complementary sequence substantially at least 5 nucleotides in length to a portion of the at least one primer extension sequencing reaction product.

[0008] Using the method of the present invention, primer extension sequencing reaction products can be directly purified without the time-consuming concentration or precipitation steps typically required in current DNA sequencing protocols. In addition, by using the method of the present invention, by selecting an appropriate capture probe, it is possible to select primer extension sequencing reaction products obtained in a plurality of primer extension sequencing reactions performed in the same reaction mixture. it can. That is, the method of the present invention can shorten the preparation time required to perform a plurality of independent sequencing reactions by performing many primer extension sequencing reactions collectively.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides both methods and devices used to purify a test sample comprising a target molecule. The use of the singular term "target molecule" is used only for simplicity in this application, and includes the plural form of "target molecule". I do. The methods described herein use a nucleic acid molecule (eg, oligonucleotide) capture probe immobilized within an electrophoretic medium. The capture probe may be dispersed in the electrophoretic medium, or may be immobilized in different layers of the medium as described in U.S. application Ser. No. 08 / 971,845. All teachings are incorporated herein by reference in their entirety. This electrophoresis medium is contained in a purification device such as a microtiter plate. The capture probe can be designed to specifically interact with and hybridize with a target molecule contained in the test sample. A test sample containing a target and a molecule other than the target can be introduced into a device, for example, a microtiter plate containing an electrophoresis medium containing an immobilized capture probe. An electric field can be applied to the purification device such that charged molecules in the test sample move through the electrophoresis medium toward an appropriate electrode. For example, in the case of the device used in Example 1, the capture voltage falls in the range of 0.1 to 200 V, more preferably between 50 and 100 V. Usually, the molecule of interest has a negative charge and moves towards a positively charged electrode. The target molecule continues to move until it contacts an immobilized capture probe specific for that particular target molecule. A hybridization complex can then be formed between the immobilized capture probe and the target molecule (
(See FIG. 1). The hybridization complex prevents further movement of the target molecule and continues movement of molecules other than the target molecule, thereby purifying the target molecule contained in the test sample. Non-target molecules include proteins such as enzymes,
Examples include small molecules such as salts, non-target nucleotides, and other non-target molecules, ie, molecules that are not targeted for further processing (see FIG. 2). Subsequently, a sufficient voltage or temperature can be applied to release the target molecule from the capture probe and exit the gel for further analysis. For example, using the apparatus used in Example 1, the target molecule can be eluted from the capture layer (that is, the layer in the electrophoresis medium containing the immobilized capture probe) at a voltage of 250 V or more. The elution voltage is 250-1000 V, more preferably 250-30.
It is preferable to be in the range of 0V. Suitable voltages for capture and elution using the purification apparatus described herein can be readily determined by one skilled in the art. Examples 4 and 5 provide methods for measuring the temperature at which target molecules are released from capture probes of various lengths.

[0010] The method of the present invention uses a purification device comprising three regions. The first area contains a test sample receiving section that receives a given test sample. The test sample receptacle can be located such that it is proximal to at least one orifice that allows delivery of a test sample (eg, a reaction mixture from a primer extension sequencing reaction). In one embodiment, the orifice is an opening at the top of the microtiter well. The second region of the purification device contains a medium for electrophoresis. The electrophoresis medium preferably contains a capture probe immobilized in the medium. This second region is preferably located physically adjacent to the first region. In one aspect, the second region is located essentially at the location of the first region and is adjacent to the first region. In a preferred embodiment, the second region is formed in one or more microtiter wells, while the first region remains capable of receiving and storing a test sample.
The third region of the purifier may be physically continuous or attached to the second region of the purifier. The third region can house a chamber capable of collecting molecules emanating from the second region, in which case the chamber is referred to as a collection chamber. The chamber can also perform other functions, such as incorporating a buffer. The refining device can also have capacity to attach to or connect to a power source (eg, a battery) that produces a DC voltage.

In one embodiment, the purification device is a multi-well set, eg, 6, 12,
Microtiter plates containing 48, 96, or 384 wells. The well or wells of the microtiter plate contain the three regions described above for the purification device. The first region includes a test sample storage section for storing a test sample. The second region contains an electrophoretic medium containing an immobilized capture probe. A third area can be formed by cutting out the bottom support of the well to create an orifice, and in some cases the microtiter well can be cut to create an opening. With chips. The bottom orifice can be coated to support the gel layer (second region) with a porous membrane if desired. Preferably, the porous membrane has a molecular weight cut-off value of greater than 15,000 daltons. The molecular weight cutoff is about 3
More preferably, it is 2,000 daltons. Preferably, the porous membrane has negligible binding of nucleic acid molecules. The bottom opening can be used to ensure access to a third area that can be physically attached to or detached from the well itself. The third region can include a collection chamber that can include a buffer.

A method for purifying a primer extension sequencing reaction product from a primer extension sequencing reaction using the purification apparatus described in the present specification is specifically included in the present invention. The target molecule is one or more primer extension sequencing reaction products formed at a particular stage of the DNA sequencing protocol. Typically, a primer extension sequencing reaction product can have a size from about 20 to about 2000 nucleotides in length. For example, a DNA molecule destined for nucleotide sequencing is
It can be located on a suitable sequencing vector such as the M13 phage vector.
Under suitable conditions well known to those skilled in the art, extended nucleic acid products can be produced using this vector, preferably using cycle sequencing (see FIG. 1; Carothers, Biotechniques, 7: 494-499 (1989), and Murr
ay), Nucleic Acid Res., 17: 8889 (1989)). Some of the reactants used in this primer extension sequencing reaction are DNA polymerase, primers, deoxynucleotides, and appropriate salts. Those skilled in the art are familiar with standard DNA sequencing protocols (Ausbel, FM, et al. Eds., Current
Protocols in Molecular Biology, vol. 1, ch. 7 (1995)). Subsequently, the target molecule (primer extension sequencing reaction product) can be subjected to purification using a purification device.

[0013] Another aspect of the invention is a kit for purifying a primer extension sequencing reaction. The kit includes an electrophoresis medium having one capture probe or a set of capture probes. At least one capture probe has a sequence at least 5 nucleotides in length that is substantially complementary to a portion of the at least one primer extension sequencing reaction product. The kit may include a sample container and a collection chamber. In one embodiment, the sample reservoir and collection chamber are located at opposite ends of the electrophoresis medium.

[0014] In a further aspect, the kit includes a plurality of electrophoresis media for purifying a plurality of primer extension sequencing reactions. The electrophoretic media can be separated from each other. Each electrophoretic medium in the kit contains one capture probe or set of capture probes. At least one capture probe in each medium has a sequence at least 5 nucleotides in length that is substantially complementary to a portion of the at least one primer extension sequencing reaction product. Each electrophoretic medium can contain the same capture probe or set of capture probes. This type of kit can be used to purify reaction products from the same primer extension sequencing reaction performed on multiple samples in separate reaction vessels. Alternatively, each electrophoretic medium in the kit can have a different capture probe or set of capture probes. This type of kit can be used to purify multiple primer extension sequencing reactions performed in the same reaction vessel.

If the kit contains more than one electrophoresis medium, each medium can be separated from other electrophoresis media in the wells of a microtiter plate.
Each electrophoretic medium can be attached to a separate sample compartment and a separate collection chamber.

In one embodiment of the invention, the target molecule is used simultaneously with both the first end (eg, at or near the 5 ′ end) and the second end (eg, at or near the 3 ′ end) of the DNA template. A method for purifying a plurality of primer extension sequencing reaction products (primer extension sequencing reaction products) formed by synthesis is described. Primer extension sequencing occurs simultaneously in both directions of the template. In this embodiment, the first terminal primer and the second terminal primer are simultaneously annealed to the template DNA, and extension in both directions is possible using one template DNA. A primer extension sequencing reaction then occurs, producing a primer extension sequencing reaction product generated from both the first and second ends of the DNA template. The primer extension sequencing reaction product can then be added to a purification device that can purify the target molecule based on whether the synthesis used a first or second terminal primer.

In this aspect, the purification device includes at least two electrophoresis gel cartridges that can be integrated to form a continuum between the two cartridges. A gel cartridge is a device that can house and support an electrophoretic medium. The gel cartridge includes an electrophoretic medium that includes a capture probe immobilized within the medium. However, each cartridge contains an electrophoretic medium containing a different immobilized capture probe. For example, one cartridge is
An electrophoresis medium comprising an immobilized capture probe comprising a nucleotide sequence substantially identical to the nucleotide sequence adjacent or adjacent to the first end of the template (capture probe "A"); The second cartridge may comprise an electrophoretic medium comprising an immobilized capture probe comprising a nucleotide sequence substantially identical to the nucleotide sequence adjacent or adjacent to the second end of the template (capture probe " B "). "Substantially identical" means greater than 70% sequence identity and / or
Or nucleotide sequences having similarity (eg, 75%, 80%, 85%, 90%, or 95% or more homology). An initial search for substantially identical nucleotide sequences was performed using the BLAST network service at the NCBI in the GenBank (release 87.0), EMBL (release 39.0), and SwissProt (release 30.0) databases. Can be done against Altshul, SF, et al., Basic
Local Alignment Search Tool, J. Mol. Biol., 215: 403 (1990); the disclosures of this document are all incorporated herein by reference. Computer analysis of nucleotide sequences was performed using MOTIFS and Genetics Computing Group.
This can be done using the FindPatterns subroutine of the p (GCG, version 8.0) software. Nucleotide comparisons can also be made according to the Higgins and Sharp method (Higgins, DG and PM
Sharp), Description of the Method used in CLUSTAL, Gene, 73: 237-244 (1
998)).

The two cartridges can be arranged such that the target molecule can move from one cartridge to the next.

For example, a cartridge with an “A” capture probe is initially positioned to receive a sample, and a second cartridge with a “B” capture probe is configured to receive a sample that has moved from the first cartridge. Put in position. A test sample containing a heterogeneous mixture of target molecules (synthesized using a first end primer and a second end primer) is added to the purification device, and then the target molecule is synthesized. The target molecule can be purified or separated based on the primers used for the purification. When an electric field is applied to this purification device, the target molecules in the test sample move by electrophoresis. Target molecules using the first end primer for synthesis are captured in the first cartridge containing "A" (a suitable capture probe) as the capture probe, whereas those target molecules using the second end primer are , Past the first cartridge and captured in a second capture cartridge containing a "B" capture probe (a suitable capture probe). After performing the electrophoretic transfer, the cartridge may be detached and placed in a separate collection chamber, such that the first and second terminal primer target molecules are separately recovered. it can.

In the method of the present invention, an electrophoresis substrate suitable for electrophoresis can be used. Suitable substrates include acrylamide and agarose, both of which are widely used for nucleic acid electrophoresis. However, other materials may be used. Examples include chemically modified acrylamide, starch, dextran, cellulose-based polymers, and the like. Other examples include modified acrylamides and acrylates (eg, Warringto, PA).
n, PA) from Polymer Sciences, Inc. (Polymer & Monomer catalo).
g. ) 1996-1997), starch (Smithies, Bioch).
em. J. 71: 585 (1959); St. Louis, MO (St.
(Louis, MO) Sigma Chemical Company
o. ) Product number S5651), dextran (e.g., Polymer Monomer Catalog 1996-19 from Polyscience, Inc., Warrington, PA).
97, and cellulose-based polymers (eg, Quesad).
a, Current Opin. in Biotechnology, 8:82
-93 (1997)). Any of these polymers listed above can be chemically modified to specifically attach capture probes for use in the present invention.

[0021] The capture probes of the present invention are generally nucleic acids, modified nucleic acids, or nucleic acid analogs. A capture probe is complementary to a primer extension sequencing reaction product, but not to a primer for sequencing primer extension. Bind nucleic acids,
Methods for making nucleic acid-containing gels are known to those skilled in the art. Nucleic acids, modified nucleic acids, and nucleic acid analogs can be attached to agarose, dextran, cellulose, and starch polymers using cyanogen bromide activation or cyanuric chloride activation. The polymer containing a carboxyl group, using a carbodiimide bond,
It can be attached to a synthetic capture probe having a primary amine group. Polymers with primary amines can be attached to amine-containing probes by glutaraldehyde or cyanuric chloride. Many polymers can be modified with thiol-reactive groups that can bind to thiol-containing synthetic probes. Many other suitable methods are found in the literature. (For an overview, see Wong, “Chemistry of Protein Co.
njugation and Cross-linking ", CRC Press, Boca Raton, Florida, 199.
(See 3)

Various capture probes can be used in the method of the present invention. Generally, a capture probe of the invention comprises a nucleic acid (eg, a nucleotide) having a nucleotide sequence substantially complementary to a nucleotide sequence region contained within the target molecule, wherein the target molecule hybridizes to the capture probe. Soy. It is important to note that the capture probe is not complementary to the primer used in the primer extension sequencing reaction. The complementarity of the capture probe to the target molecule is sufficient as long as it specifically binds to the target molecule and purifies the target molecule in the reaction mixture. Probes suitable for use in the present invention include RNA, DNA,
There are nucleic acid analogs (such as PNA), modified nucleic acids, and nucleic acids having another organic component, such as mixed species chimeric probes that include peptide nucleic acids (PNA). Capture probes can be single-stranded or double-stranded nucleic acids. Generally, the length of the capture probe is at least 5 bases in length, more typically from 5 to 50 bases, but may be thousands of bases in length.

Methods have also been developed for covalently attaching a capture probe as described herein to a polymerizable chemical group. A matrix containing a high concentration of immobilized nucleic acid can be prepared by copolymerization with an appropriate mixture of polymerizable monomer compounds. An example of a method of covalently attaching a nucleic acid to a polymerizable chemical group is described in US patent application Ser.
U.S. Patent Application Serial No. 08 / 971,845; and Rehman.
, F. N. Et al., Nucleic Acid Res. , 27: 649-655 (
1999), the teachings of which are incorporated herein by reference.

In some methods, it may be useful to use a composite matrix that includes a mixture of two or more matrix-forming substances. One example is a composite acrylamide-agarose gel. These gels generally contain 2-5% acrylamide and 0.5-1% agarose. In these gels, acrylamide provides the main sieving function, but without agarose, the concentration of the acrylamide gel is too low and lacks physical strength for convenient handling. Agarose provides physical support without significantly altering the sieving properties of acrylamide. In this case, it is preferred that the nucleic acid can bind to the component that imparts the sieving function of the gel, because the component most closely contacts the nucleic acid target in the liquid phase.

For many applications, gel-forming matrices such as agarose and cross-linked polyacrylamide are preferred. However, when applied to capillary electrophoresis (CE), it is convenient and reproducible to use a soluble polymer as the electrophoresis matrix. An example of a soluble polymer that has been found to be useful for CE analysis is Quesada (Current Opinion. In Bi).
polyacrylamide, poly (N, N-dimethylacrylamide), poly (hydroxyethylcellulose), poly (ethylene oxide) and linear chains of poly (vinyl alcohol) as described in O. Technology, 8: 82-93 (1997). It is a polymer. These soluble matrices can also be used to carry out the method of the invention. To prepare a soluble polymer matrix containing an immobilized capture probe, a “polymerizable complex containing nucleic acid (Nucleic Acid-Containing Plymerizable) is used.
It is particularly convenient to use the method described in US patent application Ser. No. 08 / 812,105 entitled “eComplex”. Another method of attaching nucleic acid molecule probes to preformed polyacrylamide gels is described by Timofevev et al., N.
ucleic Acids Res. , 24: 3142-3148 (1996).
Which can also be used to attach capture probes to prepolymerized soluble linear polyacrylamide.

It is also possible to attach the nucleic acids to particles which themselves can be incorporated into an electrophoretic matrix. The particles may be macroscopic, fine, or colloidal. (Warrington, P
A, 1995-1 from Polysciences, Inc.
996 particle catalog). Cantor et al., US Pat. No. 5,482,863, describe a method of casting an electrophoretic gel containing suspensions and particles. Using methods similar to those described above, the particles are bound to the nucleic acids, mixed with the gel-forming compound, and cast into a template as a suspension to the desired matrix shape (cast).
).

As defined herein, the term “nucleic acid” refers to DNA (deoxyribonucleic acid)
Or RNA (ribonucleic acid). A nucleic acid that is referred to herein as "isolated" refers to a nucleic acid that has been separated from components that are its source (e.g., those present in a cell or a mixture of nucleic acids, such as a library). And may be further processed. Isolated nucleic acids include those obtained by methods known to those of skill in the art. These isolated nucleic acids include substantially pure nucleic acids, nucleic acids produced by chemical synthesis, and nucleic acids produced by a combination of biological and chemical methods, as well as isolated sets. There is a modified nucleic acid and the like.

Here, the “nucleic acid analog” includes a nucleic acid containing a modified sugar group, phosphate group, or modified base. Examples of nucleic acids having modified bases include, for example, acetylated bases, carboxylated bases, or methylated bases (eg, 4-acetylcytidine, 5-carboxymethylaminomethyluridine, -Methylinosine, norvaline, or allo-isoleucine). Such nucleic acid analogs are known to those skilled in the art. One example of a useful nucleic acid analog is
A standard nucleotide base is a peptide nucleic acid (PNA) attached to a modified peptide backbone consisting of repeating N- (2-aminoethyl) glycine units (N
Ielsen et al., Science, 254: 1497-1500 (1991)).
. This peptide backbone can keep the bases at the proper distance so that they base pair with the normal DNA and RNA single strands. Presumably, the fact that the PNA strand lacks a negatively charged phosphodiester bond makes the PNA-DNA hybrid duplex much longer than the equivalent DNA-DNA duplex. Furthermore, PN
A is very resistant to nuclease degradation due to its unique structure. For these reasons, PNA nucleic acid analogs are useful in immobilized probe assays. It will be apparent to one of skill in the art that similar design methods can be used to construct other nucleic acid analogs with properties useful for immobilized probe assays.
Probes containing modified nucleic acid molecules are also useful. For example, deazaguanine (deaz
Nucleic acid molecules containing aguanine and uracil bases can be used in place of guanine and thymine containing nucleic acid molecules to reduce the thermal stability of the hybridized probe (Wetmur, Critical rev)
eyes in Biochemistry and Molecular B
ology, 26: 227-259 (1991)). Similarly, if it is desired to increase the thermostability of the hybrid, 5-methylcytosine can be used instead of cytosine (Wetmur, Critical review).
ews in Biochemistry and Molecular Bi
ology, 26: 227-259 (1991)). Modification of the sugar group of ribose, such as addition of a 2'-O-methyl group, can reduce the nuclease sensitivity of the immobilized RNA probe (Wagner, Nature, 372).
: 333-335 (1994)). Modifications that remove the negative charge from the phosphodiester backbone can increase the thermal stability of the hybrids (Moody et al., Nu.
cleic Acids Res. , 17: 4769-4782 (1989);
Iyer et al. Biol. Chem. , 270: 14712-14717 (1
995)).

Here, “substantially complementary” means that the nucleic acid molecule sequence of the capture probe does not need to accurately reflect the nucleic acid molecule sequence of the target molecule of the microorganism, but the identity of the sequence is
This means that it must be sufficiently similar to hybridize to the target molecule under certain conditions. For example, if a sequence has bases that are sufficiently complementary to hybridize, non-complementary bases or additional nucleic acid molecules may be dispersed throughout the sequence. Generally, the degree of complementarity when using short capture probes (about 20 nucleotides in length) is greater than about 95%. About 15 to about 2
With successive segments that are complementary to each other and 0 nucleotides in length, the longer the probe, the significantly lower the degree of complementarity required.

The specific conditions for carrying out the hybridization can be determined empirically by those skilled in the art. For example, conditions for stringency must be selected that significantly reduce non-specific hybridization reactions. The stringency conditions for performing nucleic acid hybridization are described, for example, in the latest protocol of molecular biology (Current Protocols in M).
olecular Biology), Ausubel, F. et al. M. Hen, Vol.
. , Suppl. 26, 1991; the teachings of which are incorporated herein by reference in their entirety. Factors such as probe length, base composition, percent mismatch between hybridizing sequences, temperature, and ionic strength
Affects nucleic acid hybrid stability. For example, stringent conditions, such as moderate or high stringency, can be determined empirically, depending on some of the properties of the probe and the microbial target molecule.

The features and other details of the invention will be more specifically described below and made clear by way of example. It is to be understood that certain aspects of the invention are presented by way of example and not limitation. The main features of the present invention are:
It can be used in various aspects without departing from the scope of the invention.

Examples The following examples do not limit the scope of the invention, but merely teach specific and practical means for carrying out the invention.

Example 1 Gel-Based DNA Isolation and Elution The sequencing reaction product was a −21M13 forward primer (5′-dye 1-spacer-TGT ^* AAAACGACGGCCAGT-3 ′ [SEQ ID NO: 1]).
Prepared using a ThermoSequenase® DYEnamic direct cycle sequencing kit with ^* Is one of the four fluorescent energy transfer dyes, as specified by the manufacturer (Amersham Pharmacia Biote)
ch, Piscataway, NJ). Each of the four reactions was performed with 1 mL of M13mp18 single-stranded DNA (0.25 m
g / mL, New England BioLabs catalog # 404-C),
Prepared by mixing 14 mL of distilled water and 2 mL of the manufacturer's reaction mixture. Each of the four reactions includes a different primer labeled with a dye called “dye 1”, “dye 2”, “dye 3” and “dye 4”, and a different dd
A mixture of NTP nucleotides was used. Next, these four reaction tubes were connected to a thermal cycler (PTC100, MJResearch, Watertown, M
A) and subjected to 30 cycles of 95 ° C. for 30 seconds, 45 ° C. for 15 seconds and 70 ° C. for 30 seconds. The four reactants were then pooled (total volume 100
mL), 11 mL of loading buffer (2.5% wt / vo in a total volume of 10 mL of distilled water)
1 xylene cyanol, 2.5% wt / vol bromophenol blue, 2
0 mM EDTA, pH 8.0, average molecular weight of 15% (wt / vol) Ficoll, 400,000) was added.

The polyacrylamide gel for electrophoretic hybridization purification was
Standard micropipette tip for 200 μL micropipette (Gilson P
200, Fisher Scientific, Pittsburgh, PA
(A yellow chip manufactured by Fisher Corporation). For the purification step, two gel chips were overlaid so that the sequencing reaction could be sequentially subjected to electrophoresis in one step through each chip (see FIG. 4A). The gel (10) in the upper chip was washed with 1 × TBE buffer (89 mM Tris-borate pH 8.3, 2 mM EDT).
A (Bio-Rad), 20 μL of 5% polyacrylamide gel (29
: 1 monomer: bis wt / wt). This upper gel is intended to capture high molecular weight M13 template DNA that provides little electrophoretic mobility under the conditions used to capture the extension sequencing reaction product. Upon removal of the high molecular weight template, Molecular Dynamics (Su
A capillary electrophoresis device such as Megabase (nnyvale, Calif.) improves the quality of the sequencing results.

The gel of the lower chip (20) contains an immobilized nucleic acid molecule-capturing probe (5′-acrylamide-GGG ATC CTC TAG AGT CGA CCT3 ′ [SEQ ID NO: 3]) at a concentration of 10 μM (for nucleic acid molecular chains). Same as the gel on the top chip except for inclusion. This capture probe is complementary to the sequence in the extension product immediately 3 'to the sequencing primer, as shown in FIG.

As shown in FIG. 3, the cloned insert to be sequenced is located 5 ′ to the illustrated template portion. Thus, as shown, the capture probe is complementary to the extended sequencing reaction product, but not to the sequencing primer.
Thus, electrophoresis of the extension sequencing reaction product on the gel of the lower chip
The extension product can be captured by hybridization without preventing excess primer from passing through the chip in electrophoresis.

The capture probe was modified with a 5′-acrylamide group using acrylamide phosphoramidite (Acrydite®, Mosaic Technologies. Boston, Mass.). Probes were immobilized on a gel matrix by adding the unpolymerized acrylamide mixture and copolymerizing directly in the gel tip.

The tips were overlaid as shown in FIG. 4A, with the probe-containing tip (20) on the bottom. Not only the area above the upper gel chip (40) but also the area between the two chips (30) is electrophoresis buffer (89 mM Tris-borate, pH 8.0).
3,2 mM EDTA, 1xTBE). Immerse the lower tip (10) in a 1.5 mL microcentrifuge tube (50) filled with buffer. A separate platinum electrode (
60) is placed in the buffer on the gel in the top tube and the buffer in the centrifuge tube (50). The upper electrode is connected to the negative lead of the power supply, and the lower electrode is attached to the positive lead.

The top chip of the device shown in FIG. 4A was loaded with 75 μL of the pooled sequencing reactions in 15 μL aliquots every 10 minutes for 1 hour. Meanwhile, the chip was subjected to electrophoresis at a voltage of 100 V during the loading process. An electric field is applied for an additional 3 hours to ensure that all sequencing reaction products are captured on the gel in the lower chip. Primers, nucleotides and excess salts that are not complementary to the immobilized capture nucleic acid molecule probe in the lower gel pass through the gel toward the lower tube.

After electrophoresis, the upper gel chip containing the slow-migrating template was discarded, and then the lower gel chip (20) was placed in the second device depicted in FIG. 4B. The lower end of the chip has a membrane (75) that cuts off the molecular weight of 3000 Dalton (Microc
on3, Amicon / Millipore, Bedford, Mass.) in an electrophoresis buffer in an ultrafiltration device (70) with a bottom surface. The ultrafiltration unit is partially immersed in a microcentrifuge tube (80) filled with 1xTBE containing a positively charged platinum electrode (60). The negatively charged electrode (60) is immersed in a buffer solution (1 × TBE) on the gel of the chip. An ultrafiltration membrane was used to prevent the eluted sequencing reaction products from migrating to the electrodes. At the electrode, the product is damaged by the electrochemical reaction. A 300 V electric field was applied to the device for 3 minutes to elute the sequencing reaction products. This voltage was sufficient to elute the sequencing reaction product from the capture probe of the gel and concentrate the product in the ultrafiltration unit.

FIG. 5 shows the effect of changing the elution voltage. The sequencing reaction products were captured and purified by electrophoretic hybridization capture as described above.
The chip is then subjected to the indicated electrophoresis conditions, and then the fluorescence imaging device (F
fluoroimager 595, Molecular Dynamics,
(Sunnyvale, CA) to visualize the fluorescent sequencing reaction products. As can be seen, voltages above 250 V completely elute the fluorescent sequencing reaction product.

To characterize the eluted product, samples of purified and crudely purified sequencing reaction products were electrophoresed in a polyacrylamide gel having different layers of capture probes immobilized on the gel as horizontal bands full gel width. Electrophoresis (see “Capture layer” in FIG. 6). The gel was 5% polyacrylamide (29: 1 monomer: bis wt / w
t) Consisting of 1xTBE. The capture layer included 10 μM 5′-acrylamide capture probe (5′-acrylamide-GGG ATC CTC TAG AGT CGA CCT3 ′ [SEQ ID NO: 3]) in addition to the same polyacrylamide and buffer. The samples were subjected to electrophoresis at 150 V for 30 minutes (FIG. 6A) and 60 minutes (FIG. 6B). Lane 1 contains 15 μL of sample purified by electrophoretic capture and elution, and Lane 2 contains 5 μL of unpurified sequencing reaction product.
Wrap. FIG. 6A shows that the hybridization-purified product (lane 1) was purified from excess primer found in the crude sample at the bottom of lane 2.

Example 2 Purification of a Single DNA Product Complementary to the M13mp18 Sequence FIG. 7A provides a schematic illustration of a method and apparatus for purifying and optionally enriching the products of the DNA sequencing reactions described below. Show. FIG. 7B shows a photograph of the capture probe-gel-chip after electrophoresis and before elution of the captured nucleotide sequence.

FIG. 8 shows a photograph of a gel showing the results of purifying the desired oligonucleotide sequence from a DNA sequencing reaction containing primers, salts, DNA template, unincorporated nucleotides, and a dye terminator. . First, the DNA sequencing product was purified by a gel-loaded chip to obtain a crude sample, and then the crude sample was purified by a capture probe-gel-chip. Lane 1 shows the pattern obtained before purification. Lane 2 shows the pattern seen after purification using a gel-loaded chip. Removal of the DNA template has been demonstrated. Lane 3 shows the capture probe.
The pattern seen after purification using a gel-chip is shown. The lack of a DNA template reveals the localization of the desired sequence.

A. Preparation of Separation Devices With and Without Immobilized Capture Probes Preparation of separation devices containing Acrydite® oligonucleotide gels was performed as follows. Hybrigel solution was converted to 40% acrylamide (
29: 1 acrylamide monomer: bis-acrylamide) and 10% TB
E (90 mM Tris-Borate-EDTA buffer pH 8.3; reagents are from Biorad Laboratories. Inc .; Hercules, C
(Purchased from A). The Acrydite® oligonucleotide for the capture probe was replaced with the oligonucleotide (Operon Tech).
nologies, Inc. And Acrylamide phosphoramidite (Acrydite polymer is available from Mosaic Tec);
hnologies, Inc. US Pat. No. 5,641,658; and Kenney, Ray, and Bo.
les, BioTechniques 25, 516-521 (1998). The individual disclosures are all incorporated herein by reference). Hybrigel has 5% acrylamide (29: 1), 1 × TBE, and 10 (M Acrydite® having the following sequence:
An oligonucleotide capture probe [SEQ ID NO: 4] was included. 5'-acrylamide-GCT GAG ATC TCC TAG GG3 '[
SEQ ID NO: 4] The capture probe selected in this example has a sequence that is complementary to a part of the polylinker of the vector M13mp18 that provides a DNA sequencing reaction product that is the target molecule. Does not contain any primer sequences. 10% ammonium persulfate and N, N, N'N'-tetramethylethylenediamine (TE
(MED; BioRad, Hercules, Calif.) To the Hybrigel solution accelerated the polymerization (within 2 minutes).

To prepare the probe-gel-tip, 1 μm of 10% ammonium persulfate
1 and 0.5 μl of TEMED were added to 200 μl of the Hybrigel solution. A solution containing 10 μl of polymerized Hybridel oligonucleotide is quickly added to 2 μl.
Pipette to a 00 μl tip and polymerize. Probe-gel-chip
Eight or twelve were made at a time using a multipipette device. For storage, the probe-gel-tip was squirted into an Eppendorf tube containing about 0.3 ml of 1 × TBE. Care must be taken that the gel does not move off the chip. The probe-gel-chip was then overlaid with 150 μl of 1 × TBE.

A gel-loaded chip was used to remove template DNA. Acrylamide (
The gel-loaded chip was prepared to contain only 29: 1) (ie, no Acrydite capture probe). The gel-load-tip is forty as described above.
% Acrylamide (29: 1 monomer: bis) and a solution containing 5% acrylamide (29: 1), 1 × TBE formed from a stock solution of 10 × TBE. 10% Ammonium persulfate and TEMED were added to the solution, and 200 μl of the final solution was pipetted to each tip. Gel-load-tips can also be stored as described above.

B. Preparation of DNA sequencing reaction product and capture probe PE-Applied Biosystems (PE-Applied Biosystems)
) The sequencing reaction product was prepared using the following PE Applied Biosystems Big Dye Primer Cycle Sequencing Kit (PE Applied Biosy)
stems BigDye Primer Cycle Sequencer
g Kit) (available from PE-Applied Biosystems), prepared with the -21 M13 forward primer on GeneAmp2400 using cycling conditions recommended by PE Applied Biosystems. The vector M13mp18 was used. A portion of DNA having a known sequence was inserted after the primer site. An extension product was prepared. A capture probe was created in the region between the primer and the inserted DNA.
A capture probe capable of hybridizing to the extension product from the forward primer was selected. The capture probe was Acrydite® at the 5 ′ end.
And oligonucleotides synthesized using

Alternatively, Amersham-Pharmacia Dynamic ET
・ Terminator cycle sequencing kit (DYEnamic E)
It is also possible to use T Terminator Cycle Sequencing Kit.

C. Capture of Extension Products for DNA Sequencing Electrophoretic capture and separation of selected extension products (targets in test samples) was performed as follows. 10 μl of the sequencing reaction solution containing primers, salts, unincorporated nucleotides, dye terminator, and template DNA (ie,
１ ／ of one reaction) and the target DNA extension product synthesized from the template DNA
l of 10x Ficoll loading buffer (35% Ficoll 400, 0.1% bromophenol blue, 0.1% xylene cyanol, 100 mM EDTA)
To obtain a sample solution. This sample solution was laminated to the gel surface in a gel-load-tip (ie, without the Acrydite capture probe) to remove template DNA. This chip was overlaid on a probe-gel-chip, a Hybridel-containing chip, as shown in FIG. 7A. A small amount of electrophoresis buffer was layered on the gel surface in the probe-gel-chip and the gel-load-chip was placed in contact with the electrophoresis buffer. Therefore, when the electrodes and power were turned on, a liquid connection was made between the two gels that allowed an electrical connection.

After loading the sample solution on the gel surface of the gel-load-tip, both tips were
Placed in Eppendorf tubes containing xTBE electrophoresis buffer. Platinum electrodes were placed in the electrophoresis buffer above and below the stacked gel chips and
Was applied for 10 minutes. The upper electrode was connected to the negative lead of the power supply, and the lower electrode was attached to the positive electrode of the power supply. The gel-loaded chip was used to introduce the partially purified sample solution into the probe-gel-chip. Smaller DNA fragments pass through the buffer and then migrate into the probe-gel-chip faster than the DNA template and dye retained on the gel-loaded chip.

The electrophoresis buffer remaining on the gel surface of the probe-gel-chip was removed.
The electrophoresis buffer was replaced with 4 μl of formamide-loaded dye (5: 1 deionized formamide, 25 mg / ml blue dextran, 25 mM EDTA). Raising the gel temperature in the probe-gel-chip to 55 ° C. and placing an Eppendorf tube containing the lower buffer reservoir in Dry Bath (VWR) facilitated separation of the target oligonucleotide sequence from the capture probe. . A clean second gel-loaded chip using a 30% acrylamide gel containing 5% acrylic acid was lowered to enter the formamide loaded dye. A platinum electrode was placed in the top gel-load tip. The current direction was reversed to push the oligonucleotides dissociated from the hybridization complex upward. One minute at 40 V was sufficient to push the oligonucleotide off the probe-gel-chip and into the formamide-loaded dye. 4 μl of sample was removed with a pipette and retained for sequence analysis. After the electrophoresis, the gel in the probe-gel-chip was subjected to Molecular Dynamics Fluorimager.
595 was used for visualization. The results shown in FIG. 7B indicate that capture occurs at the upper surface of the gel in the probe-gel-chip.

D. Analysis of Sequencing Purification by Hybridgel Assay Glass Plates (10 × 10 cm, 0.75 m
m), and the sandwich is reduced to about half by 20% acrylamide (29: 1; Bio-Rad), 1 × TBE (90 mM Tri).
s-borate buffer, pH 8.3, 2 mM EDTA). Polymerization was performed by adding 10% aqueous ammonium persulfate solution (APS) and TEMED to each of 1
Started by adding at gel volumes of / 100 and 1/1000. For a gel with one capture layer, a final concentration of 600 μl gel solution containing 10 μM Acrydite-labeled oligonucleotide (20% polyacrylamide, 1 ×
TBE, 4 μl of 10% APS and 4 μl of 10% TEMED) were polymerized. After polymerization of the acquisition layer, the remaining space in the plate sandwich was filled with 5% gel. The composite gel was then mounted in a minigel device containing 1xTBE and subjected to electrophoresis at 100V-150V for about 45 minutes. After electrophoresis, Mol
Using e.g. dynamic Dynamics Fluorimager 595,
The gel was visualized. This result proves that the sequence captured by the hybrid gel probe is the complement of the template DNA.

E. Automatic Sequencing of Captured Oligonucleotides The oligonucleotide sequence purified by the device of the present invention was analyzed by an automatic sequence analyzer according to the above steps. The accuracy of the decision proved always to be better than 99%. The readable sequence reaches at least 750 nucleotides.

Example 3: Simultaneous Separation of Various DNA Sequencing Reaction Products This example demonstrates that the method of the present invention is useful for sequencing plasmid-replicated oligonucleotide inserts. is there. As shown in FIG. 9A, forward and reverse primers are used. Plasmids with large inserts were sequenced simultaneously using two primers in one reaction vessel. Two probes each containing a different capture probe-gel-
The chips were placed in series. The probe sequence was designed based on the forward and reverse primers used. The slab gel shown in FIG. 9C demonstrates the purification of two oligonucleotide targets in the test sample compared to the crude product. Noteworthy is that the sequence of the reverse product (right) indicates that it is not contaminated with the forward sequence. It should also be noted that contaminants of the DNA template have been removed.

A. Preparation of Separation Devices With and Without Immobilized Capture Probes Gel-loaded tips were prepared as described in Example 2. The probe-gel-chip was prepared as described in Example 2, but as shown in FIG. 9A, one was provided with a forward-direction primer probe and the other was provided with a reverse-direction primer probe. Thus, two separate hybrid gel probes-gel-
The chip was made such that one had an acridite oligonucleotide (SEQ ID NO: 5) and the other had an acridite oligonucleotide (SEQ ID NO: 6). Plasmid p698, a plasmid vector pGEM3Zf (-) (Promega Biotech, WI) having a 3.8 kb insert at the Xbal site of the polylinker, was used. The two probes are Xbal in the polylinker.
It is derived from the sequence on either side of the site. SEQ ID NO: 5 is complementary to the sequencing reaction product generated using the reverse primer and is therefore captureable. Similarly, SEQ ID NO: 6 is capable of capturing a sequence from the forward primer. (SEQ ID NO: 5) 5 'acrylamide-TGCAGGCATGCAAGCTT (SEQ ID NO: 6) 5' acrylamide-GGGTACCGAGCTCGAATT
C

Thus, each oligonucleotide primer probe has an A at the 5 ′ end.
It is synthesized using cydite and can be hybridized to the extension product. Each capture probe sequence is complementary to both the extension product sequence and the primer. Each capture probe sequence is specific for a particular vector derived from the region between the primer site and the insert DNA.

B. Purification of Multiple Reaction Products As shown in FIG. 9A, a probe 2-gel-chip containing SEQ ID NO: 6 is placed in series (overlap) with a probe 3-gel-chip containing SEQ ID NO: 5. did.
A running electrophoresis buffer was placed on the upper surface of the probe 3-gel-chip to provide electrical contact to prevent drying. Test samples from multiple sequencing reactions were electrophoresed through both probe 2-gel-chip and probe 3-gel-chip. FIG. 9B shows capture by two distinct probes on two separate chips.

Example 4: Elution of Target from Capture Probe Using Temperature Gradient The stability of the hybridization complex is temperature dependent. A vertical slab gel was prepared that included a layer of Acrydite® capture probe sandwiched between gel layers without the capture probe. The gels for the upper and lower layers were used for gel-load-tip. The Acrydite probe layer was prepared as described for the probe-gel-chip using the capture probe sequence (SEQ ID NO: 5) in Example 2. The sample in this case was a fluorescent oligonucleotide having a sequence complementary to SEQ ID NO: 5. The same sample was loaded into each well. The entire gel was subjected to a temperature gradient using an aluminum backplate and two water baths. The gel temperature on the left is 23 ° C. The temperature rose to 53 ° C on the right across the gel. At low temperatures, the target was efficiently captured on top of the capture layer. As the temperature increased, target capture was inhibited by the time the sample passed through the layer (see the gel image in FIG. 10). The transition temperature, ie, the temperature at which the target is no longer captured, is related to Tm, but was not the only factor known to be included alone.

Example 5: Dependence of Sequence Elution Temperature and Capture Probe Size To determine the temperature conditions for capture and elution of the sequencing reaction product, the experiment shown in FIG. 11 was performed. This experiment demonstrates that the elution temperature is affected by the size of the capture probe. Elution temperature was shown to be affected by the size of the capture probe. The five panels shown in FIG. 11 show two identical slab gels.
Shown are migrations from 3 ° C. to 5 different temperatures, where 5 different capture probes were used. The number of bases in each probe sequence was 13, 15, 17, 19 and 21 nucleotides as shown in FIG. 11 (from left to right). Each lane was loaded with an M13 sequencing reaction (Dynamic® ET). 1
The 3-mer capture probe did not efficiently capture the sequencing reaction product at 23 ° C. All other capture probes (ie, having lengths 15, 17, 17 and 21) captured the sequencing reaction products at 23 ° C. At 30 ° C., the 15-mer released the sequence, at 35 ° C. the 17-mer began to release, and at 50 ° C., all target sequences were released. These results indicate the temperature conditions for capture and elution of the sequencing reaction product.

The 17-mer Acrydite probe was selected as the capture probe in a standard procedure where elution at 45 ° C. occurred.

Equivalents Although the invention has been shown and described in detail with reference to the preferred embodiments of the invention, it is to be understood that the invention may be configured without departing from the spirit and scope of the invention as defined by the appended claims. It is understood by those skilled in the art that various changes in detail and details are possible.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a method for purifying a target nucleic acid molecule from an extension sequencing reaction using an electrophoresis gel having a capture probe immobilized within the gel region.

FIG. 2 is a schematic diagram of steps involved in a method of purifying an extension product using a microtiter well containing an electrophoresis medium containing a capture probe immobilized in the medium. . In FIG. 2, “ddNTPs” represents dideoxynucleotide triphosphate, “pol” represents DNA polymerase, and “extension product (ex.
ducts) "represents the product of the primer extension sequencing reaction.

FIG. 3 shows the results of the forward primer (SEQ ID NO: 1) and template (SEQ ID NO: 1) used in Example 1.
It is a block diagram of SEQ ID NO: 2) and a capture probe (SEQ ID NO: 3).

FIG. 4 is a schematic diagram illustrating an experimental design for DNA isolation using an electrophoresis medium.

FIG. 5 shows the effect of changing the elution voltage.

FIG. 6 shows the results obtained by subjecting the extension sequencing reaction product to electrophoresis in which the electrophoresis medium comprises an immobilized capture probe. FIG. 6A shows the experimental results after running the gel for 30 minutes. FIG. 6B shows the experimental result after 60 minutes.

FIG. 7A is a schematic diagram of an apparatus and a method used for purifying a primer extension sequencing reaction product of Example 2. FIG. 7B shows the distribution of primer extension sequencing reaction products in the lower gel-chip comprising the capture probe.

FIG. 8 shows images before purification (lane 1), after purification using only the upper gel-chip (lane 2), and after purification using the lower gel-chip comprising the capture probe (lane 3). FIG. 4 is a photograph of an electrophoresis gel showing the distribution of components of the primer extension sequencing reaction performed. Lanes 4-6 contain various concentrations of purified M13 DNA.

FIG. 9A is a schematic diagram of the forward and reverse primer extension sequencing reactions performed simultaneously in Example 3. FIG. 9B is an image of the forward and reverse primer extension sequencing reaction products separated using two gel-chips. The upper gel-chip contains a capture probe that is complementary to the product of the forward primer extension sequencing reaction.
The lower gel-chip contains a capture probe that is complementary to the reverse primer extension sequencing reaction product. FIG. 9C is an image of the separation of the products of the forward and reverse primer extension sequencing reaction products on a slab gel that contained two capture probes (lane 1).
). Lane 2 shows the purity of the reverse primer extension sequencing reaction product after separation from the forward primer extension sequencing reaction product. One capture probe was complementary to the reaction product of the forward sequencing reaction and the other capture probe was complementary to the reaction product of the reverse sequencing reaction. FIG. 9D is an analysis result of the reverse sequencing reaction product after purification by the method of the present invention.

FIG. 10 is a temperature gradient gel used to measure the temperature at which a target is released from a capture probe.

FIG. 11 is an image of a gel used to measure the temperature at which a target is emitted from capture probes of various lengths.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ware, Lawrence USA Massachusetts 01748 Hopkinton, Granite Street 55 (72) Inventor Daranda, Raul, Kay. United States Massachusetts 02143 Somerville, Linden Avenue Number Two 27 (72) Inventor Summers, Nevin, M. United States Massachusetts 02446 Brooklyn, Monmouth Street 75 F-term (Reference) 4B024 AA20 CA01 CA09 FA18 HA14 4B029 AA07 AA23 BB20 CC03 CC08 FA12 4B063 QA13 QQ42 QR32 QR55 QR62 QS25 QS34

Claims (27)

[Claims] (A) introducing a primer extension sequencing reaction mixture into a purification device containing an electrophoresis medium containing an immobilized capture probe; (b) electrophoresis in step (a) Subjecting the medium to an electric field to cause electrophoretic migration of one or more target molecules to at least one region of the electrophoretic medium comprising an immobilized capture probe to which the target molecules bind, (c) A method for purifying a target molecule from a primer extension sequencing reaction using a purification device, comprising: collecting the target molecule in step (b). 2. The method according to claim 1, wherein the purification device is a microtiter plate. 3. The method of claim 2, wherein the microtiter plate contains a plurality of wells. 4. The method according to claim 1, wherein the number of wells contained in the microtiter plate is 6, 1
4. The method according to claim 3, wherein the method is selected from the group consisting of 2, 48, 96 and 384. 5. In step (c), a voltage is applied sufficient to release the target molecule from its complementary capture probe, and the target molecule continues to move electrophoretically under the influence of the electric field, 2. The medium exiting the medium and collecting in a collection chamber.
The described method. 6. The method according to claim 5, wherein the polarity of the electric field is reversed, and the released target molecule moves back to the test sample receiving portion and is subjected to collection. 7. The method according to claim 1, wherein the capture probe is a nucleic acid molecule. 8. The method of claim 7, wherein the capture probe is complementary to a primer extension sequencing reaction product. 9. The method of claim 8, wherein the capture probe is about 20 to about 2000 nucleotides in length. 10. (a) Unique of immobilized capture probe
Introducing the primer extension sequencing reaction mixture into a purification device containing at least two cartridges each containing an electrophoresis medium containing a suitable set, (b) using the electrophoresis medium of step (a). Subjecting to an electric field to cause electrophoretic migration of one or more primer extension sequencing reaction products to at least two cartridges each comprising an electrophoresis medium, wherein each medium is an immobilized capture probe Wherein the primer extension sequencing reaction product binds to an appropriate immobilized capture probe, and (c) collecting the target molecule of step (b). Multiple primer extension sequencing formed by synthesizing a primer extension sequencing reaction product using both of the second ends Method of purifying the reaction product. 11. The method according to claim 10, wherein the purification device is a microtiter plate. 12. The method according to claim 11, wherein the microtiter plate contains a plurality of wells. 13. The method according to claim 1, wherein the number of wells contained in the microtiter plate is 6,
13. The method of claim 12, wherein the method is selected from the group consisting of 12, 48, 96, and 384. 14. In step (c), a voltage is applied sufficient to release the target molecule from its complementary capture probe, and the target molecule continues to move electrophoretically under the influence of an electric field, The method of claim 10, wherein the medium exits and collects in a collection chamber. 15. The method according to claim 14, wherein the polarity of the electric field is reversed, and the released target molecule moves back to the test sample receiving portion and is subjected to collection. 16. The method according to claim 10, wherein the capture probe is a nucleic acid molecule. 17. The method of claim 16, wherein the capture probe is complementary to a primer extension sequencing reaction product. 18. The method of claim 17, wherein the capture probe is between about 20 and about 2000 nucleotides in length. 19. An electrophoresis comprising a capture probe or set of capture probes having a complementary sequence substantially at least 5 nucleotide bases in length as part of at least one primer extension sequencing reaction product. A kit for purifying a primer extension sequencing reaction product, comprising a medium for use. 20. The kit according to claim 19, further comprising a test sample storage part and a collection chamber at the opposite end of the electrophoresis medium. 21. The kit of claim 20, wherein the capture probe is about 20 to about 2000 nucleotide bases in length. 22. A plurality comprising a capture probe or a set of capture probes each having a complementary sequence substantially at least 5 nucleotide bases in length in a portion of at least one primer extension sequencing reaction product. A kit for purifying a plurality of primer extension sequencing reactions, comprising a medium for electrophoresis. 23. The kit according to claim 22, wherein the electrophoresis medium is separated into wells of a microtiter plate. 24. The sample load storage section and the sample collection chamber each further comprising a plurality of test sample storage sections and a plurality of collection chambers located at opposite ends of the electrophoresis medium. Kit. 25. The kit of claim 24, wherein the capture probe is about 20 to about 2000 nucleotide bases in length. 26. The kit of claim 23, wherein each electrophoretic medium comprises the same capture probe or set of capture probes. 27. The kit of claim 23, wherein each electrophoretic medium comprises a different capture probe or set of capture probes.