JP2003531389A - MIPC chromatography apparatus with improved temperature control - Google Patents

Abstract

(57) Abstract: A liquid chromatography apparatus having appropriate stationary phase and mobile phase temperature control for polynucleotide separation by MIPC and DMIPC methods. The apparatus comprises a heater means (80) having a temperature control system; a matched ion polynucleotide chromatography separation column (72) having an inlet end (70); a single coil capillary having an inlet end and an outlet end (68). including. The outlet end of the capillary is connected to the inlet end (70) of the separation column (72). The inlet end of the capillary (68) contains means for receiving the process liquid, the tube has a length of 6 to 400 cm and has a linear tube length heating means. The coils of the separation column (72) and the capillary (68) are sealed in a heater means (80). The capillary is preferably PEEK or titanium. The heater means (80) can be an air bath oven. Preferably, it is a heat conductive block.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION [0001] The present invention is herein distinguished by Matched Ion Polynucleotide Chromatography (Matched) to distinguish it from traditional partitioning-based reverse phase HPLC systems.
Ion Polynucleotide Chromatography) (M
IPC) for high performance chromatographic separation systems. In particular, the invention is
It relates to a MIPC chromatography system with an improved high precision column heater system.

BACKGROUND OF THE INVENTION Traditional chromatography is a separation method based on the partitioning of mixture components between a "stationary phase" and a "mobile phase". The stationary phase is provided by the surface of a solid material which can comprise a number of different materials in the form of particles or passage surfaces such as cellulose, silica gel, coated silica gel, polymeric beads, polysaccharides and the like. These materials can be maintained on a solid surface such as on a glass plate or packed in a column. The mobile phase can be a liquid or a gas in gas chromatography. The present invention relates to liquid mobile phases.

Separation principles are generally the same regardless of the material used, the form of the material, or the equipment used. Different components of the mixture have different respective solubilities in the stationary phase and in the mobile phase. Thus, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are distributed between the stationary and mobile phases. As the mobile phase passes through the column, the equilibrium constantly shifts predominantly to the mobile phase. This occurs because the equilibrium mixture will always recognize and partition the new mobile phase. As the mobile phase is carried down the column, it recognizes and partitions into the new stationary phase. After all, there was no more stationary phase at the end of the column,
The sample simply exits the column in the mobile phase.

Separation of the mixture components occurs because the mixture components have slightly different affinities for the stationary phase and / or solubility in the mobile phase and therefore different partition equilibrium values. Therefore, the mixture components travel down the column at different rates.

It is known that one way to improve the separation is to increase the surface area of the stationary phase, since the chromatographic separation depends on the interaction with the stationary phase. The best separation has less interaction, ie a low partition coefficient and a longer column,
Obtained when giving increased interaction.

In traditional liquid chromatography, a glass column is packed with stationary phase particles,
The mobile phase is then pulled through the column only by gravity. However, when smaller stationary phase particles are used in the column, the force of gravity alone is insufficient to cause the mobile phase to flow through the column. Instead, pressure must be applied. However, glass columns can only withstand about 200 psi. Passing the mobile phase through a column packed with 5 micron particles requires applying a pressure of about 2000 psi or higher to the column. Currently, 5-10 micron particles are the standard. Particles smaller than 5 microns are used for particularly difficult separations or in some special cases. This method is designated by the term "high performance liquid chromatography" or HPLC.

HPLC allows the use of much more different types of particles used to separate a much wider variety of chemical structures than was possible with large particle gravity columns. However, the separation principle remains the same.

HPLC-based ion-pairing chromatography methods have recently been introduced to effectively separate mixtures of double-stranded polynucleotides in general, and DNA in particular, where the separation is based on base pair length (Bonn ( U.S. Pat. No. 5,585 to 1996).
236; Huber, et al. , Chromatographia 37
: 653 (1993); Huber, et al. , Anal. Biochem
． 212: 351 (1993)). All of these references and the references contained therein are incorporated herein in their entirety. The term "matched ion polynucleotide chromatography" (MIPC) has been applied to this method by applicants as the understanding of the mechanism of DNA separation advances. MIPC is D
NA fragments are separated based on base pair length and are not constrained by the deficiencies associated with gel-based separation methods.

Matched ion polynucleotide chromatography, as used herein, is defined as a method for separating single-stranded and double-stranded polynucleotides using a non-polar separation medium, where the method is a paired method. An ionic factor and an organic solvent that releases the polynucleotide from the separation medium are used. Basic MIPC isolation is completed in less than 10 minutes and often less than 5 minutes. For more difficult separations, such as those using isocratic solvent conditions, the separation time will be longer. Effective isocratic and targeted compartment elution requires tight control of separation conditions.

As the use and understanding of MIPC has evolved, MI is partially denatured, ie, at a temperature sufficient to denature heteroduplexes at sites of base pair mismatches.
PC analysis was performed and found to be able to separate homoduplexes from heteroduplexes having the same base pair length (US Pat. No. 5,795,976; Hayward).
-Lester, et al. , Genome Research 5: 494
(1995); Underhill, et al. , Proc. Natl. Ac
ad. Sci. USA 93: 193 (1996); Doris, et al.
, DHPLC Workshop, Stanford University,
(1997)). All of these references and the references contained therein are incorporated herein in their entirety. Therefore, the use of denaturing HPLC (DHPLC) has been applied to mutation detection (Underhill, et al., Genom.
e Research 7: 996 (1997); Liu, et al. , Nu
cleic Acid Res. , 26; 1396 (1998)).

DHPLC can separate heteroduplexes that differ by as little as one base pair. However, the separation of homoduplexes and heteroduplexes cannot be fully analyzed. Artifacts and impurities can also interfere with the interpretation of DHPLC separation chromatograms in that it can be difficult to distinguish artifacts or impurities from putative mutations (Underhil).
1, et al. , Genome Res. 7: 996 (1997)). The presence of mutations may even be completely overlooked (Liu, et al.,
Nucleic Acid Res. 26: 1396 (1998)). The references cited above and the references contained therein are incorporated herein in their entirety.

The accuracy, reproducibility, convenience and speed of DHPLC-based DNA fragment separation and mutation detection assays have been compromised so far due to problems associated with DHPLC systems. DNA by HPLC and DHPLC that has not been studied so far
Important aspects of separation and mutation detection include treatment of material comprising chromatographic system components; treatment of material comprising separation medium; method development times (methods).
solvent pre-selection to minimize development time; optimum temperature pre-selection to bring about partial denaturation of the heteroduplex during MIPC:
And optimization of DHPLC for automated high throughput mutation detection screening assays. These factors are essential for obtaining clear, accurate, reproducible and high throughput DNA isolation and mutation detection results.

Therefore, it is possible to separate DNA fragments based on their size differences in an accurate, reproducible and reliable way and also DNAs of the same length but with different base pair sequences ( HPL which can also isolate mutations from wild type)
There is a need for a C system. Such a system should be automated and efficient, should be adaptable to routine high throughput sample screening applications, and provide high throughput sample screening with minimal operator attention. Should be.

The basic MIPC separation method differs from the traditional HPLC separation method in that no separation is performed by a series of equilibrium separations between mobile and stationary phases as the liquid passes through the column. Instead, the sample is applied to the column with a solvent strength that allows the sample dsDNA to bind to the surface of the separation medium. Strands of specific base pair length are irreversibly removed from the stationary phase surface by specific solvent concentrations and carried down the column. By passing increasing gradients of solvent through the sample, even larger base pair lengths are successfully removed in succession and passed through the column. When the DNA is released from the stationary phase, its linear velocity rapidly reaches that of the mobile phase. The basic separation does not depend on column length or stationary phase area.

The isocratic variation of the MICP method is a DNA having the same size.
It can be applied to separate a mixture of fragments, where the fragments in the mixture show different degrees of non-polarity.

The application of matched ion polynucleotide chromatography (MIPC) under partially denaturing conditions used to separate heteroduplexes from homoduplexes in mutation detection is referred to below as DMIPC.

Separation of double-stranded deoxyribonucleic acid (dsDNA) fragments and detection of DNA mutations are of great importance in medicine, in physical science and sociology, and in forensic investigations. The Human Genome Project provides a vast amount of genetic information and provides new information for assessing the association between mutations and human disease (Guyer, et al., Proc. Natl. Acad.
Sci. USA 92: 10841 (1995)). For example, the underlying cause of disease is indicated by a genetic code that differs from wild type (Cotton, TIG 13: 4).
3 (1997)). Understanding the genetic basis of the disease can be a starting point for treatment. Similarly, determination of differences in the genetic code can provide powerful and perhaps clear insights into evolutionary and population studies (Cooper, e.
t al. , Human Genetics vol. 69: 201 (1985
)). Understanding these and other issues with the genetic code requires the ability to identify abnormalities, or mutations, in a DNA fragment relative to the wild type.

DNA molecules are polymers that comprise subunits called deoxynucleotides. The four deoxynucleotides present in DNA are common cyclic sugars,
Deoxyribose, which is herein referred to as A, G, C, respectively.
And covalently bound to any of the four bases referred to as T, adenine (purine), guanine (purine), cytosine (pyrimidine) and thymine (pyrimidine). The phosphate group links the 3'-hydroxyl of one deoxynucleotide with the 5'-hydroxyl of another deoxynucleotide to form a polymer chain. In double-stranded DNA, the two strands are organized into a helical structure by so-called hydrogen bonds between complementary bases. The complementarity of bases is determined by their chemical structure.
In double-stranded DNA, each A is paired with T and each G is paired with C, ie the purine is paired with pyrimidine. Ideally, DNA is replicated in exact copy by a DNA polymerase during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by the polymerase chain reaction (PCR).

Occasionally, exact replication fails and incorrect base pairing occurs. Further replication of the new strand is a double-stranded DN containing a heritable difference in the base sequence from the parental one.
Generate A descendants. Such heritable mutations in the base pair sequence are called mutations.

As used herein, double-stranded DNA is referred to as double-stranded. When the base sequence of one strand is completely complementary to the base sequence of the other strand, the double strand is called a homoduplex. A duplex is said to be a heteroduplex if it contains at least one base pair that is not complementary. Heteroduplexes are formed during DNA replication when the DNA polymerase enzyme makes an error, and non-complementary bases are added to the replicated polynucleotide chain. Further replication of the heteroduplex ideally produces homoduplexes that are heterologous, that is, these homoduplexes have altered sequences as compared to the original parental DNA strand. . If the parental DNA has a sequence that predominates in the naturally occurring population, that sequence is commonly referred to as "wild-type."

Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, "point mutations" or "single base pair mutations" that result in one incorrect base pairing. The most common point mutations comprise a "transition" in which one purine or pyrimidine base is replaced by another and a "transversion" in which the purine is replaced by a pyrimidine (and vice versa). . Point mutations also comprise mutations in which one base is added or removed from the DNA strand. Such "insertions" or "deletions" are also known as "frameshift mutations". Although occurring less frequently than point mutations, larger mutations affecting multiple base pairs can also occur and may be significant. A more detailed description of mutations can be found in US Pat. No. 5,459,0 to Modrich (1995).
39 and US Pat. No. 5,698,400 to Cotton (1997). All of these references and the references contained therein are incorporated herein by reference in their entireties.

The sequence of base pairs in DNA is the code for the production of proteins. In particular, the DNA sequence in the exon portion of the DNA chain encodes the corresponding amino acid sequence in the protein. Therefore, mutations in the DNA sequence may result in alterations in the amino acid sequence of the protein. Such a modification in the amino acid sequence may be entirely benign, or it may inactivate the protein or modify its function to be life-threatening or lethal.
On the other hand, the intron part does not contain the code for protein production, so DNA
Mutations in the intron portion of the chain are not expected to have biological effects. Nevertheless, mutation detection in the intron portion can be important, for example, in forensic investigations.

Therefore, detection of mutations is of great importance in diagnosing a disease, understanding the origin of the disease, and developing potential treatments. Also, the detection of mutations and the identification of similarities or differences in DNA samples is a method in increasing global food supply by developing crop varieties that are disease resistant and / or have higher yields. It is of decisive importance in science, in evolutionary and population studies, and also in scientific research in general (Guyer, et al.
al. , Proc. Natl. Acad. Sci. USA 92: 10841 (
1995); Cotton, TIG 13:43 (1997)).

Modifications in DNA sequences that are benign or have no negative consequences are sometimes referred to as “polymorphisms”. For the purposes of this application, all modifications in a DNA sequence, whether they have negative consequences or not, are defined herein as "mutations." For brevity, the term "mutation" is used herein to mean a modification in the base sequence of a DNA strand relative to a reference strand (generally, but not necessarily, wild type). As used herein, "
The term "mutation" includes the term "polymorphism" or any other similar or equivalent term in the art.

Prior to the present invention, size-based analysis of DNA samples was accomplished by standard gel electrophoresis (GEP). Capillary gel electrophoresis (CGE) has also been used to separate and analyze mixtures of DNA fragments having different lengths, eg, digestion products resulting from restriction enzyme digestion of DNA samples. However, these methods show that DNs having the same base pair length but different base sequences are used.
The A fragment cannot be distinguished. This is a serious limitation of GEP.

Mutations in heteroduplex DNA strands under “partially denaturing” conditions are based on gel-based analytical methods such as denaturing gradient gel electrophoresis (DGGE) and denaturing gradient gel capillary electrophoresis (DGGC). Can be detected by. The term "partially denatured" refers to DNA in which the other parts of the duplex are left intact, ie not separated.
It is defined as the separation of mismatched base pairs (due to temperature, pH, solvent or other factors) in the duplex. The phenomenon of "partial denaturation" occurs because heteroduplexes denature at sites of base pair mismatches at the lower temperatures required to denature the rest of the chain.

These gel-based techniques are difficult and require highly skilled experimental scientists. Moreover, each analysis requires very long preparations and separations. Denaturing capillary gel electrophoresis analysis can only be performed on smaller fragments. Separation of 90 base pair fragments takes more than 30 minutes. Gradient denaturing gels run overnight and require about a day of set up time. A further defect in the gradient gel is the adaptation of these methods to isolate isolated DNA fragments (which requires specialized techniques and equipment) and the conditions required for isolation. It is difficult to do. Conditions must be experimentally developed for each fragment (Laboratory Methods for the Detectio).
no of Mutations and Polymorphisms, ed.
G. R. Taylor, CRC Press, 1997). The long analysis times of gel methodologies are further exacerbated by the migration of DNA fragments in the gel being inversely proportional to the length of the DNA fragments in a geometric relationship. Therefore, the longer D
The analysis time for NA fragments can often be falsified.

In addition to the deficiencies of the denaturing gel method described above, these techniques are always reproducible or accurate because performing gel preparation and analysis can be very diverse from operator to operator. Not necessarily.

Separation of the double stranded nucleic acid fragment mixture by GEP or DGGE produces bands in a linear arrangement, each band in the arrangement corresponding to a separated double stranded nucleic acid component of the mixture. Typically, multiple mixtures are simultaneously separated and analyzed in separate lanes on the same gel slab, thus producing a parallel series of such linearly arranged bands. Bands are often curved rather than straight, their mobility and shape can vary across the width of the gel, and lanes and bands can mix with each other. The source of such inaccuracies arises from the lack of uniformity and homogeneity of gel beds, electroosmosis, thermal gradients and diffusion effects, and numerous other factors. This type of inaccuracy is well known in the GEP art and can lead to significant distortion and inaccuracy in the display of separation results. Moreover, the band display data obtained from GEP separations are not quantitative or accurate due to uncertainties regarding band shape and integrity. Even if the linear band arrangement is scanned with a detector and the resulting data is integrated, the linear band arrangement is scanned only over the center of the band, thus providing an accurate quantification of the linear band arrangement display caused by GEP separation. I can't do it. Since the detector only captures a small fraction of any given band and the bands are not uniform, the results produced by the scanning method are not accurate and can even be misleading.

Methods of visualizing GEP and DGGE separations, such as staining or autoradiography, are also cumbersome and time consuming. Furthermore, the separation data is in hard copy form and cannot be stored electronically for easy information retrieval and comparison, nor can it be enhanced in image quality to improve visualization of dense separations.

This MIPC method is temperature sensitive and, for example, the MIPC separation method described in co-pending US patent application Ser. No. 09 / 129,105, filed Aug. 4, 1998, is more stringent. Good temperature control is especially important. Strict temperature control is performed at a partially denaturing temperature, that is, at a temperature at which a mismatched DNA existing at a mutation site of a heteroduplex is denatured but a matched DNA remains bound to a double-stranded phase. Required to keep both stationary phases.

In many prior art HPLC applications, the HPLC separation method can be carried out at room temperature. However, MIPC, especially DMIPC, is used at high temperature while thermostat-controlling the temperature of the column. Prior art HPLC temperature controls were generally able to set and keep the temperature within a range of ± 1 ° C and reach equilibrium temperature within 10 minutes. Temperature control did not provide the accuracy or precision required by the MIPC method. The system did not result in defining and keeping the column and solution temperature at the same value.

In the advanced MIPC and DMIPC methods, the temperature control within ± 0.1 ° C.
It is necessary to reach equilibrium at the new set temperature within minutes. Moreover, automatic oven control must provide the specific oven conditions required for each particular method or separation. Maintaining a constant temperature is particularly important in quantitative analysis, as changes in temperature can seriously affect peak size measurements. These devices usually consist of a high speed air blower and an electronically controlled thermostat in a similar arrangement to that used in gas chromatographs. Alternatively, the LC column can be coated and the temperature can be controlled by a contact heater or by circulating the liquid from a constant temperature bath. Although this latter method is practical for routine analysis, it does improve the time response requirements of the improved high capacity system of the present invention,
Does not meet accuracy or precision.

SUMMARY OF THE INVENTION MIPC, in which both the temperature of the column and the solution introduced into the column are carried out
It is an object of the present invention to provide a column heating system which keeps at the same exact and exact temperature, which is a feature required by the DMIPC separation method.

There are both methods, such as the temperature of the column and the solution introduced into the column, without loss of precision or accuracy as required for some methods, eg, experimental thermotitration to identify the optimal denaturing MIPC temperature. It is another object of the present invention to provide a column heating system that can quickly change from a temperature value to another.

In summary, the present invention is a liquid chromatography device with stationary phase and mobile phase temperature control suitable for polynucleotide separation by MIPC and DMIPC methods. It comprises a heater means having a temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; and a capillary having an inlet end and an outlet end. The outlet end of the capillary is connected to the inlet end of the separation column, and the inlet end of the capillary comprises means for receiving the process liquid. The tube is fully extended, preferably 6-400 cm.
end length) and can be a coil. The separation column and the coil of the capillary are enclosed in a heater means. The temperature control system is preferably calibrated to the temperature of the liquid flowing into the column.

The capillaries are preferably PEEK or titanium. The inlet end of the capillary coil may lead to a prefilter, which may optionally be enclosed in a heater means.

One embodiment of the liquid chromatography device of the present invention comprises an air bath oven with a recirculating air temperature control system and a matched ion polynucleotide chromatography separation column with inlet and outlet ends. It comprises a single coil capillary having an inlet end and an outlet end, the outlet end of the capillary being connected to the inlet end of the separation column and the inlet end of the capillary comprising means for receiving the process liquid. The tube preferably has a fully extended length of 6-400 cm. The separation column and capillary coil are sealed in an air bath oven. Preferably, the inlet end of the capillary coil leads to a prefilter, which is sealed in an air bath oven.

[0039] Preferably, the device air bath system includes a recirculation air temperature control system comprising a temperature sensor, a heater and a heater control. The temperature sensor is located in the air bath oven in the path of the recirculating air and is connected to the heater control. A heater control is coupled to the heater and the heater and the heater control comprise means for adjusting the temperature of the recirculated air in the air bath.

The invention also relates to the temperature of the liquid entering the column, such as infrared temperature sensor measurement or the use of a contact temperature sensor (liquid temperature sensor) arranged on the capillary connecting the column with the means for receiving the process liquid. It also includes a method of calibrating the temperature control by directly measuring Establish a table of corresponding standards for liquid temperature and air temperature. First, the user sets the system temperature. Then the corresponding air temperature is calculated from the table. Set the heater to this air temperature. The liquid temperature is then read by system control and compared to the set temperature.
If the difference between the set temperature and the liquid temperature is larger than the allowable limit, the heater setting must be adjusted by raising or lowering the heater setting depending on the difference between the set temperature and the liquid temperature. Next, the calibration table is adjusted to register the actual set temperature and the air temperature and the liquid temperature. Repeat this procedure until the difference between the liquid temperature and the set temperature is within the allowable limit.

Furthermore, a temperature sensor can be mounted directly on the coil of the capillary for a more accurate temperature reading.

In another aspect of the invention, the heater means comprises a heat conductive block having a first primary heat transfer surface, a separation column inlet and a capillary coil inlet. The separation column is placed in thermal communication with its inner wall within the separation column inlet; and the capillary coil is placed in the capillary coil inlet. The outer end of the coil is in thermal conductivity with the inner wall of the capillary outlet. The system includes a heater means, the heater means being in thermal communication with the first heat transfer surface. The system preferably includes a heater control, the heater control being coupled to the thermal sensor and the heater means. The system can include a radiator, wherein the thermally conductive block has a second heat transfer surface,
The radiator is in a heat conducting relationship with the second heat transfer surface.

Preferably the thermally conductive block comprises a thermal sensor outlet and a thermal sensor,
The heat sensor is arranged in a heat conducting relationship with the inner surface of the heat sensor insertion port.

The heating means may be a Peltier heating / cooling device, and the Peltier heating / cooling device is in thermal communication with the first heat transfer surface. The heater control is connected to the thermal sensor and Peltier heating and cooling device.

DETAILED DESCRIPTION OF THE INVENTION The present invention allows the separation of DNA fragments based on size differences by MIPC and also by DMIPC to identify the presence of mutations from homoduplex to wild type strand. An HPLC system is also provided which is capable of separating the DNA heteroduplex of the mutant strand paired with. It allows these separations to be performed in an accurate, reproducible and reliable manner.

Tight temperature control of the separation column and of the solution passing through it is partially denatured used to separate heteroduplexes from homoduplexes in optimal DNA fragment separation and mutation detection by MIPC. It is required for mutation detection by DMIPC, which is an application of matched ion polynucleotide chromatography (MIPC) under conditions.

FIG. 1 is a perspective view of an HPLC DNA analyzer system suitable for use in separating DNA fragments into size-based fractions. The system can comprise stacked components, as shown in this presentation, or it can be integrated into a single cabinet or housing. The main components are represented here by a column oven housing 2 containing a separation column and a detector housing 4 containing a detector which measures the properties of the column eluent as a function of the concentration of the substance to be separated. The autosampler housing 6 contains sample vials or well trays and conventional autosampling components. The pump housing 8 contains a pump for moving the liquid solvent, reagent solution and sample solution into the column. The degasser housing 10 houses the solution degasser. Control and system monitoring features include a control interface 11, a conventional desktop computer 12 with a CPU, an input keyboard 14 and an output monitor 16. The system is an ordinary printer,
It can be connected to a network and auxiliary storage (not shown).

FIG. 2 is a schematic diagram of a system showing the relationship between operation components. Computer control 18
Under the order of the eluent 19, the eluent 19 is pumped by the pump 20 to
Proceed through the column 22 to the column and column oven 24. Eluents include liquid solvents, reagent solutions and sample solutions required for the separation process.
Polynucleotide samples, such as DNA fragments, RNA samples and other oligonucleotides, are removed from sample containers (vials, trays, etc.) in sample source 26 and mixed with solvent solution in autosampler assembly 22. As will be shown in greater detail below, the solution and separation columns are brought to the desired controlled temperature in the column oven 24.

The separation column separates the polynucleotide components passing through it into fractions based on size, and these fractions pass through the detector 28 and to the fraction collector 30 in the eluent solution. Be done.

The system components except the oven are conventional HPLC system components readily available from domestic and foreign manufacturers. Transgenomic, Inc. (Omaha,
Commercially available WAVE DNA Fragment Analysis available from
These components are combined with specialized columns in a zer system.

For more information on MIPC and DMIPC methods, equipment and separation columns, see US Pat. Nos. 5,585,236, 5,772,889, 5,795,976 and co-pending US patent application Ser. No. 09. / 058,580, 09 / 058,337, 09 / 129,105, the entire contents of all of which are incorporated herein by reference.

FIG. 3 is a diagram of a conventional prior art HPLC DNA analyzer column oven. The housing 2 comprises a separation column 32, a prefilter 34 and a capillary section 36,
Contains 38 and 40. Separation column 32 has an inlet end 42 and an outlet end 44. The inlet end 42 is connected to the prefilter 34 by a capillary tube 38. The prefilter 34 communicates with the autosampler through a capillary tube 36. The outlet conduit 40 leads from the outlet end 44 of the column 32 to the analyzer device. Traditional HP
In the LC system, the dead volumes exhibited by the capillaries 36, 38 and 40 and the prefilter 34 reduced the separation efficiency and resulted in peak broadening. Therefore, capillary length has traditionally been kept to a minimum.

The air circulating through the housing 2 and passing over the prefilter 34, the column 32 and the capillary segments 36, 38 and 40 must fix and maintain these components and the passing liquid at a selected temperature. . The housing 2 has a wall 46
It is divided into a processing section and a heater fan section. Air exits the treatment compartment through exhaust port 48 in wall 46, is drawn by fan 50, passes through heater component 52, and returns to the treatment compartment. Outlet air temperature is monitored by a temperature sensor 54, which has a lead that interfaces with a conventional heater temperature controller 56.

If sufficient time has passed to equilibrate the temperature of the system and system components, the temperature of the tube, prefilter and column components can be kept within ± 1 ° C. by circulating air. However, air and system components have severely limited opportunities to heat the liquid entering the column. Temperature uniformity is compromised during system operation due to heat transfer to and from liquids passing through the capillaries and columns.

We have found that the MIPC system is relatively insensitive to capillaries leading from the sample injector and dead space in the separation column. In fact, dead volumes of up to a few milliliters between the sample injector and the separation column are acceptable in MIPC systems. The only constraint in dead volume is its effect on the total time required to form the mobile phase gradient. Unlike HPLC, the duality of separation is unaffected by dead space. As a result, capillaries can be lengthened without compromising column separation. This feature allows the oven modification shown in FIGS. 4 and 5. FIG. 4 is a front view of the processing compartment of an HPLC DNA analyzer column oven according to the present invention, and FIG. 5 is a top view of the HPLC DNA analyzer column oven shown in FIG.

The processing compartment in the embodiment shown in FIGS. 4 and 5 comprises a wall 5 on which an air outlet 60 is arranged.
Separated from the heating compartment by 8. A temperature sensor, such as the thermistor 62, is located at the exhaust to monitor the temperature of the air passing through the exhaust 60.

The capillary 64 leads from a sample injector (not shown) to the prefilter 66. Prefilter 66 is an inline filter or guard cartridge as described in US Pat. No. 5,772,889. It removes contaminants from the incoming liquid. Capillary extension coil (elongate)
d coil) 68 has inlet end communication with it for receiving process liquid from prefilter 66. The extension coil 68 is connected to the inlet end 7 of the separation column 72.
It has an outlet end leading to zero. Separation column 72 is described in US Pat.
236 and co-pending US patent application Ser. Nos. 09 / 058,580, 09/0
58,337, 09 / 183,123, 09 / 183,450, containing MIPC separation media. The outlet tube 74 leads from an outlet end 76 of the separation column 72 to a detector (not shown).

Coil 68 is a liquid heating coil made of DNA compatible polyvalent cation free tubing having the desired thermal conductivity. Titanium or PEEK as described in US Pat. No. 5,772,889 and application 09 / 081,040 are preferred as they can remove multivalent ion contaminants. The length and diameter of the tubing used is any length sufficient to allow the liquid passing through it to reach the equilibrium temperature of the air in the processing compartment. Usually 6-400 cm long and 0.15-0.
A tube ID of 4 mm is sufficient. The length of tubing 68 does not reduce the separation of components achieved by the system, so that the length can be selected based on the length required to effect effective heating of the process liquid.

With respect to FIG. 5, the air is heated and recirculated as described in FIG. Air from the treatment compartment 78 passes through an opening 60 in the wall 58 through a heater / fan system 80 for temperature regulation. Conditioned air received by the heating compartment 82 is recirculated to the processing compartment 78 along a passageway 84 defined by the space between the wall 58 and the oven outer wall 86.

The heating coil in the embodiment shown in FIGS. 4 and 5 provides temperature accuracy within a range of ± 0.2 ° C. and a temperature equilibration time between temperature settings of less than 5 minutes for a temperature change of 5 ° C.
Temperature changes up to 1 ° C reduce to less than 2 minutes.

FIG. 6 is a side view of a preferred compact dual column heater embodiment of the present invention, and FIG. 7 is a cross-sectional view taken along line 7-7 in FIG. This embodiment uses direct metal-to-metal heat transfer to and from the system components to achieve temperature changes and accuracy and does not rely on an air bath.

This preferred embodiment is shown for a two column system, although it could be used for a single column if desired. It comprises a thermally conductive block (88, 90) having outlets sized and shaped to contain system components. Filter holes or pre-filter insertion ports (92, 94) are pre-filters (96, 98)
And has an inner surface sized to establish heat transfer contact with its outer surface. The separation column insertion ports (100, 102) are connected to the respective separation columns (104
, 106) and a separation column coupler that connects a capillary to each separation column (
108, 110) and has an inner surface sized to accommodate. Outlet (10
0, 102) are sized and shaped to establish heat transfer contact between the internal heat transfer surfaces of the blocks (88, 90) and the separation column components contained therein. The capillary coil outlet 112 (one shown in FIG. 7) was shaped to accommodate the coil of the capillary 114 (one shown in FIG. 7) and to establish heat transfer contact with its outer surface. Has an inner surface. In the embodiment shown in these figures, the insertion ports (92, 94) and (10
0, 102) are cylindrical holes with substantially parallel central axes lying in a common plane. It will be readily apparent to those skilled in the art that other arrangements are equally suitable, and that all arrangements are considered within the scope of the invention.

The temperature sensor inlets (116, 118) are provided with heat conductive blocks (88, 90).
Provided in. Capillary outlet passages 112 for receiving the connecting tubes 122 in heat conductive relationship are also provided in the heat conductive blocks (88, 90). Capillary coil outlet 112 is shown in this figure to be a cylindrical hole with its axes perpendicular to the axes of the inlets (92, 94) and (100, 102). Optionally, in order to increase the heat transfer area between the metal block heating assembly and the liquid in the coil, a conductive metal cylinder in thermal conductive contact with its inner surface within the capillary coil (
(Not shown) can be placed.

Surface 1 of heating block (88, 90) to transfer heat to the heat conductive block
A KAPTON resistance heater or other type of heating device 124 is disposed between and in contact with 26 and 128 and in thermal conductive contact therewith. Cooling radiator (130
, 132) are opposed cooling surfaces (134, 1) of the thermally conductive blocks (88, 90).
36) is arranged in a heat conducting relationship with and removes heat from them. Cooling fan 138
And 140 are in heat removal relationship with cooling radiators 130 and 132 and are activated to facilitate heat removal from them.

The thermally conductive blocks 88 and 90, and the cooling radiators 130 and 132 are made of other thermally conductive solids such as ferrous metal or any other solid material having the required thermal conductivity. However, it is made of a material having a high thermal conductivity such as aluminum or copper. Heat pipes can also be used as cooling radiators.

Although titanium is preferred for maximum heat transfer efficiency, the capillaries can be made of DNA compatible PEEK or titanium. With this improved heat transfer,
Capillary coils can have a fully extended length as short as 5 cm, although a minimum coil length of 10 cm is preferred. Longer coils of PEEK are required to achieve the same heat transfer as titanium capillaries.

The system shown in FIGS. 6 and 7 comprises two systems of mirror images. It is readily apparent that half of the system is sufficient for a single column and is considered within the scope of the invention.

The location, alignment and spacing of the outlets is not a key feature of the present invention. Any alignment and arrangement that provides compact and heat transfer efficient results is considered within the scope of the present invention. Preference is given to blocking heat transfer from the casing to heating and cooling devices.

The embodiment of the invention shown in FIGS. 6 and 7 is more sensitive to heater control, resulting in a rapid change from one temperature reference to another, and temperature accuracy of ± 0.5 ° C. of set temperature. Provide a compact heater to keep within. The heat transfer rates obtained with metal-to-metal contact between the heating block and the components to be heated are much greater than those obtainable in air bath systems, resulting in faster response to changing temperatures and greater temperature accuracy. . It also allows process liquid temperature regulation with shorter capillary coils.

FIG. 8 is a schematic diagram of a Peltier heater. With the use of semiconductors in the Peltier component, relatively high temperature differences can be achieved. Each Peltier component is made of a p- or n-type semiconductor linked to a copper bridge. In this figure, component 144 is a conventional p-type doped semiconductor, such as silicon, and component 146 is a conventional n-type doped semiconductor. Components 148, 150 and 152 are copper bridges. Copper bridge 148 is cooled and copper bridges 150 and 152 are heated using a negative voltage on the p-doped side and a positive voltage on the n-doped side. When the voltage is reversed, copper bridge 148 is heated and copper bridges 150 and 152 are cooled. Thus, with a simple voltage change, the copper bridge 148 can be a source of both heating and cooling that quickly and closely regulates the temperature of the metal block in thermal conductivity therewith.

FIG. 9 is a schematic diagram of a preferred Peltier heater / cooler embodiment of the present invention. The heating block 154 is in conductive contact with a Peltier heating component (not shown) for the heating or cooling needed to reach and maintain the desired temperature.
The groove 156 is a prefilter receptor having an inner surface 158 in thermal conductive relationship with the prefilter 160. The groove 162 is the coupler 164 of the separation column 168.
And a column guard column having an inner surface 163 in thermal conductivity with the end nut component 166. Capillary 170 is in communication with prefilter 160 and sample and solution source (not shown). The capillary 174 from the outlet of the separation column 168 leads to an analyzer (not shown). Capillary 172 connects the outlet end of prefilter 160 to coupler 164, which simultaneously separates column 168.
It leads to. Capillary 172 is provided with a labyrinth-like arrangement of grooves in heating block 164 to provide increased capillary length and surface contact between capillary 172 and heating block 154. The maze arrangement can be any arrangement that provides suitable capillary length and surface contact, including annular loops and more than one row of capillary arrangements per groove.

Capillary 172 can be PEEK or titanium, with titanium being preferred due to its high thermal conductivity. The heating block 154 can be any thermally conductive metal. Ferrous metals such as steel can be used, but aluminum or copper are preferred due to their higher thermal conductivity.

The temperature sensor 175 is located in the center of the block in the corresponding receptor groove.

The Peltier heater is a Peltier thermocycler (thermocycler).
Controlled with conventional temperature and control systems (not shown) such as the system used in s). The temperature accuracy achieved by this Peltier heating block is ± 0.5 ° C.

FIG. 10 is a schematic diagram of a process liquid temperature sensor. The temperature sensor 176 is mounted on the capillary 178 with a layer of thermally conductive grease 180 between the sensor 176 and the outer surface 182 of the tube 178. The capillaries are preferably titanium. The sensor 176 is further sealed to the insulating sleeve 184. The electrical lead wire 186 is
Sensor 176 is coupled to temperature control (not shown). Placing the temperature sensor 176 directly on the outer surface of the capillary 178 increases the accuracy of temperature measurement of the liquid flowing through the tube. A layer of thermally conductive grease between the sensor 178 and tube 178 increases the area of thermal contact between the sensor and tube and further improves the accuracy of temperature readings.

The temperature sensor of FIG. 10 was also installed in the air bath oven by comparing the data from the process liquid temperature sensor with the data from the temperature sensor located in the air bath oven and calculating the deviation between the two. It can also be used to calibrate a temperature sensor.

The temperature sensor 62 shown in FIG. 4, the sensors located at the receptors 116 and 118 shown in FIG. 6, and the temperature sensor 175 shown in FIG. 9 are optimally calibrated to the temperature of the liquid passing through the capillary and separation column. .

The present invention is further described in the following specific but non-limiting examples.
In these examples, the method described in the past tense in the past tense is practiced in the laboratory. The present tense method is not practiced in the laboratory and is legally reduced to practice in the present application.

Example 1 Transgenomic Inc. The WAVE ™ DNA Fragment Separation System from E.co. Thermocycler, DNASe
Preheating line made of p ^TM column and PEEK tube (150 cm x 0.33 mm ID)
Was modified to contain. A column in which preheat tubes are wrapped around each other between PCR tube wells (ie, physically placed around the wells themselves and in thermal contact with a 96-well heating block), and then placed in holes machined from the thermocycler. Connected to. The oven response was fast, requiring about 10 seconds to reach the set temperature. It took about 2 minutes for the liquid to reach the set temperature. This response is shown in FIGS.
It was much faster than the air bath oven. The oven was cooled and heated with a Peltier device, so that the increase and decrease in temperature was brought quickly.

While the foregoing describes certain aspects of the present invention, it should be understood that these aspects are provided by way of example only. Variations to the above, but not departing from the spirit and scope of the invention as described and claimed herein, are contemplated and contemplated to be practiced.

[Brief description of drawings]

FIG. 1 is a perspective view of an HPLC DNA analyzer system suitable for use in separating DNA fragments into size-based fractions.

[Fig. 2]   FIG. 3 is a schematic diagram of a system showing the relationship between the operating components of a DNA analyzer system.

[Figure 3]   1 is a diagram of a conventional prior art HPLC DNA analyzer column oven.

FIG. 4 is a front view of the separation compartment of an improved HPLC DNA analyzer column oven according to the present invention.

[Figure 5]   5 is a top view of the HPLC DNA analyzer column oven shown in FIG. 4. FIG.

[Figure 6]   FIG. 3 is a side view of a compact dual column heater embodiment of the present invention.

[Figure 7]   7 is a cross-sectional view taken along line 7-7 in FIG.

[Figure 8]   It is a schematic diagram of a Peltier heater.

[Figure 9]   1 is a schematic diagram of a preferred Peltier heater / cooler embodiment of the present invention.

10 is a schematic diagram of temperature sensor components for temperature controlled calibration or for use as a temperature sensor in the oven shown in FIG.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 30/54 G01N 30/54 H 30/88 30/88 E (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, K, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Taylor, Paul Day 94301 Palo Alto Hawthorne Avenyu 248, California, USA

Claims (16)

[Claims] 1. A heater means having a temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; a length of capillary having an inlet end and an outlet end; the outlet end of the capillary being a separation column. The separation column and the coil of the capillary are connected to the inlet end, and the inlet end of the capillary comprises means for receiving the process liquid, the tube having a fully extended length of 6 to 400 cm; A liquid chromatography device for polynucleotide separation by the MIPC and DMIPC methods, which is arranged in the heater means; and the temperature control system is calibrated to the temperature of the liquid flowing into the separation column. 2. The liquid chromatography device according to claim 1, wherein the capillary is PEEK or titanium. 3. The liquid chromatography apparatus according to claim 1, wherein the inlet end of the capillary coil communicates with a prefilter, and the prefilter is sealed by a heater means. 4. An air bath oven having a recirculated air temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; a length of capillary having an inlet end and an outlet end; an outlet end of a capillary. Is connected to the inlet end of the separation column, and the inlet end of the capillary comprises means for receiving the process liquid, the tube having a fully extended length of 6 to 400 cm, and the separation column and The capillary coil is sealed in an air bath oven and exposed to the air therein A liquid chromatography device for polynucleotide separation by the MIPC and DMIPC methods. 5. The liquid chromatography device according to claim 4, wherein the capillary is PEEK or titanium. 6. The liquid chromatography apparatus according to claim 4, wherein the inlet end of the capillary coil communicates with a prefilter, and the prefilter is sealed in an air bath oven. 7. The recirculation air temperature control system comprises a temperature sensor, a heater and a heater control, the temperature sensor being located in the air bath oven in the path of the recirculation air and coupled to the heater control, the heater control being 5. The liquid chromatography device of claim 4, coupled to a heater, the heater and heater control comprising means for adjusting the temperature of the recirculated air in the air bath. 8. A recirculated air temperature control system comprising a temperature sensor, a heater and a heater control, the temperature sensor being placed in direct contact with the capillary and coupled to the heater control, the heater control being coupled to the heater, The heater and heater control comprise means for adjusting the temperature of the recirculated air in the air bath.
Liquid chromatography equipment. 9. A temperature sensor mounted on the capillary, a layer of thermally conductive grease, and an electrical lead connecting the sensor to a temperature control means, wherein the layer of thermally conductive grease is , A process liquid temperature control system disposed between the sensor and the outer surface of the tube in contact with both the outer surface and the sensor, the sensor being sealed in an insulating sleeve. 10. A heater means comprising a heat conductive block having a first heat transfer surface, a separation column insertion port and a capillary coil insertion port; in the separation column insertion port being in heat conduction with its inner wall. A separation column arranged; and a capillary of one coil arranged at the capillary coil inlet, the outer end of the coil being in heat-conducting relation with the inner wall of the capillary inlet, MIPC and DMIPC Chromatography apparatus for polynucleotide separation by the method. 11. The liquid chromatography apparatus according to claim 8, further comprising a heater in thermal communication with the first heat transfer surface. 12. The liquid chromatography apparatus of claim 9 including a heater control coupled to the thermal sensor and heater means. 13. The method of claim 8 including a heat sink, wherein the heat conductive block has a second heat transfer surface and the heat radiator is in heat conductive relationship with the second heat transfer surface. Liquid chromatography equipment. 14. The liquid chromatography apparatus according to claim 8, wherein the heat conductive block includes a heat sensor insertion port and a heat sensor, and the heat sensor is disposed in the heat sensor insertion port in a heat conductive relationship with an inner surface thereof. 15. The liquid chromatography apparatus according to claim 8, further comprising a Peltier heating / cooling device in thermal communication with the first heat transfer surface. 16. The liquid chromatography device of claim 13 including a heater control coupled to the thermal sensor and the Peltier heating and cooling device.