Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/02—Burettes; Pipettes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/14—Process control and prevention of errors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
  • B01L2300/0838—Capillaries
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N2035/00465—Separating and mixing arrangements
  • G01N2035/00564—Handling or washing solid phase elements, e.g. beads

Definitions

• the present invention is directed to a system for storage, purification and reaction of biopolymers, for example, deoxyribonucleic acid (DNA).
• the components of the invention comprise: a solid storage medium for DNA, a cannula for use with automatic fluid delivery systems for aspirating and dispensing fluids associated with a solid material, an apparatus for separation of low molecular
• the components of the system may be used together as a single system or, in some applications, independently as individual components of other systems.
• the invention is further directed to
• the sample is a heel-prick taken from an infant.
• the transport of liquid blood or frozen blood also demands temperature control and an appropriate transport system other than the regular postal system. This is true even before the question of hygiene is considered.
• problems with pathogens such as the HIV virus generally rule out the transport of any potentially infective liquid or frozen sample
• Blood dried on filter paper is a proven alternative to the above procedures and it has been shown that DNA can be extracted and isolated from dried whole blood spots in a form and in sufficient quantities for use in DNA analysis'.
• This procedure still suffers from a number of disadvantages. For example, there has been no deliberate and rapid destruction of most pathogens, and there has been no deliberate inhibition of the processes degrading the DNA other than that caused by desiccation. Slow desiccation or even a small degree of rehydration under conditions of high relative humidity will allow the growth of DNA-destroying microflora. Even in the presence of bacteriostatic agents of the type that do not denature proteins, there will be conditions that permit enzymic-autolytic breakdown of the DNA and some nonenzymic breakdown of the DNA.
• Enzymic-autolytic breakdown refers to the process whereby dying or damaged tissues, either human cells or parasite cells, activate enzymes that degrade their own components. Additionally, there is considerable difficulty desorbing very high molecular weight DNA from paper, if this is required. Surface adsorption effects can cause losses of DNA and this will cause the preferential loss of the least degraded DNA, i.e. the most desired class of DNA molecules. Thus, there is a need for safe, convenient, and minimally labor intensive means for storage of liquid DNA containing samples. A further problem in the past has been the treatment of biopolymers, for example DNA, subsequent to dry storage on filter paper. Such stored samples were typically not capable of rapid processing through automatic fluid delivery systems due to the construction of prior art pipettes.
• the solid medium on which the compound was adsorbed such as filter paper, could block the aspiration and dispensing port of the pipette preventing effective aspiration of reaction fluids.
• the need for compound removal from the storage medium not only caused the loss of compounds such as DNA, but also caused increased laboratory costs due to loss of time, increased labor and increased reagent costs. If it was desirable to leave the compound in association with the solid medium (or if the solid medium was desirable in the reaction system) automatic fluid delivery systems were inefficient due to the need for manually preventing the solid from obstructing the end of the pipette.
• a problem which can hinder the analysis and isolation of a biopolymer post synthesis is the separation of low molecular weight by-products from the desired product.
• methods for analysis and isolation of DNA amplified through PCR technology generally require removal of the reaction by-products from the amplification reaction prior to analysis. This is readily appreciated by considering a relatively new technology for DNA analysis, capillary electrophoresis. This method has been used successfully for automated analysis of DNA following PCR amplification. However, prior to capillary electrophoresis, it is essential to remove salts which are by-products of the PCR reaction mixture.
• DNA fingerprinting for forensic or medical diagnostic purposes is a typical example of this.
• identification of a specific sample for example a specific genotype, requires screening a large pool of samples. Rapid processing of the pooled samples is desirable to obtain results within a reasonable amount of time. As the sample quantity screened is increased there remains the need to maintain accurate correlation of a sample tested and the results generated for that sample.
• a cannula for use with an automated fluid delivery system.
• the cannula generally comprises: a structure including a shaft defining fluid channel; a mechanism for operatively connecting the cannula in fluid flow communication with the automatic fluid delivery system for selectively aspirating and dispensing fluid through the cannula; a primary fluid aspirating and dispensing port; and, at least one auxiliary fluid aspirating and dispensing port.
• the at least one auxiliary fluid aspirating and dispensing port is arranged, relative to the primary fluid aspirating and dispensing port, such that it is unlikely that both the primary port and the at least one auxiliary port will be blocked simultaneously with solid material in the region to which fluid is dispensed and from which fluid is aspirated, in use.
• the primary fluid aspirating and dispensing port is continuous with the at least one auxiliary fluid aspirating and dispensing port. This is provided, for example, by having one of the ports in an end of the cannula with the other connected to (or continuous with) the first port, but extending upwardly along the side of the cannula shaft.
• the ports are separated from one another and are geometrically located such that one piece of solid material is not likely to block both at the same time.
• both the auxiliary port(s) and the primary port(s) are provided by means of a porous frit positioned in an end of the cannula.
• an automatic fluid delivery system for aspirating and dispensing a fluid from a fluid reaction system associated with a solid material.
• the automatic fluid delivery system comprises a cannula as described above, in association with the flow arrangement constructed and arranged to dispense reaction fluid through the cannula.
• the flow arrangement may be, for example a pump system, or the like provided in the automatic fluid delivery system.
• the biopolymer purification arrangement comprises a polymeric gel shaped to hold a volume of fluid.
• the biopolymer reaction material to be purified is placed within the gel. Materials will diffuse at different rates through the gel. In the absence of an applied electric field, a large biopolymer such as DNA will tend to remain in the vessel for an extended period of time, and will not diffuse into the gel. Smaller materials, on the other hand, will tend to diffuse into the gel; the result being purification of the large biopolymer left within the receiving bore in the gel.
• the invention relates to a solid medium for use in the storage of DNA, particularly DNA in blood samples, and to methods which comprise the use of this solid medium.
• the invention relates to a method for storage and transport of DNA on the solid medium, as well as to methods which involve either (a) the recovery of the DNA from the solid medium or (b) the use of the DNA in situ on the solid medium (for example, DNA sequence amplification by the polymerase chain reaction).
• the DNA in blood samples (hereinafter referred to as "blood DNA”) is used for the purposes of diagnosis of genetic diseases, diagnosis and monitoring of blood-borne parasitic diseases such as malaria, the determination of paternity, and the monitoring of other unusual cell populations in the blood as can occur in some neoplasias.
• blood DNA is used to cover all sources of DNA commonly found in blood, and thus includes the DNA of the human patient from whom the blood sample was obtained, as well as the DNA in any other organisms circulating within his/her blood.
• the present invention provides a material and method for the removal of by-products from reactions producing biopolymers that is convenient to use and is amenable for automated sampling and analysis of the biopolymers. It is particulaily useful for the removal of by-products from PCR reactions prior to analysis of DNA by capillary electrophoresis in an automated process. It enables the products of multiple DNA amplification reactions to be processed at the same time, if this is required.
• the medium is relatively straightforward to produce and can be made in bulk in sizes to suit requirements.
• Figure 1 depicts an analysis described with respect to Experiment 3.2(c)(iii)
• Figure 2 is a prospective view of a preferred pipette or cannula according to the present invention.
• Figure 3 is an enlarged fragmentary cross-sectional view of a portion of the cannula shown in Figure 2.
• Figure 4 is an end view of the cannula shown in Figures 2 and 3.
• Figure 5 is an enlarged fragmentary cross-sectional view of a cannula according to an alternate embodiment of the present invention.
• Figure 6 is an enlarged fragmentary cross-sectional view, generally analogous to Figures 3 and 5, of yet a second alternate embodiment of the present invention.
• Figure 7 is a top plan schematic view of a vessel usable in purifications according to the present invention.
• Figure 8 is a cross-sectional view of the arrangement shown in Figure 7, taken generally along line 8-8 thereof.
• Figure 9 is a schematic view of a system for efficiently processing large quantity of biological samples, according to the present invention.
• a solid medium for storage of biopolymers for example DNA, including blood DNA, which comprises a solid matrix having a compound or composition which protects against degradation of the biopolymer incorporated into or absorbed on the matrix.
• the solid matrix comprises a solid support, for example, an absorbent cellulose-based paper (such as filter paper) or a micromesh of synthetic plastics material, with the biopolymer-protecting compound or composition absorbed onto the solid support.
• the solid matrix may include a suitable binder material so that the matrix is in the form of a compressed tablet or pellet, with the biopolymer-protecting compound or composition incorporated into or absorbed onto the tablet or pellet.
• the present invention provides a method for the storage of DNA, including blood DNA, which comprises applying the DNA to a solid medium.
• the solid medium of the invention comprises a solid matrix having a compound or composition which protects against degradation of DNA that is incorporated into or adsorbed on the matrix.
• the DNA-protecting compound or composition comprises uric acid, together with a weak base to convert the uric acid to a urate salt and to provide an alkaline pH between 8.0 and 9.5.
• a solid medium for storage of blood DNA which comprises a solid matrix having incorporated therein or absorbed thereon a composition comprising a weak base, a chelating agent and an anionic surfactant or detergent, and optionally uric acid or a urate salt.
• the composition is such as to impose an alkaline pH, such as a pH of between 8.0 and 9.5, on blood that is added to the matrix.
• a further aspect of the present invention is the long term storage of DNA on the solid medium of this invention, by impregnating the solid medium or encasing the solid medium in a protective material, for example a plastics material such as polystyrene.
• a protective material for example a plastics material such as polystyrene.
• the polystyrene may be subsequently removed when access to the stored DNA is required.
• the blood sample is applied as a blood spot to the solid medium of this aspect of the invention, where the components (more particularly the surfactant) will denature proteins and the majority of any pathogenic organisms in the sample.
• the blood DNA will be protected from degradation factors, such as oxidation and ultraviolet (UV) light and processes of the type described above so that the relatively stable, and denatured, dried blood sample can then be transported to a diagnostic laboratory.
• the DNA can be extracted or the DNA can be used in situ on the solid medium.
• composition used in this aspect of this invention comprises the following:
• a monovalent weak base such as "Tris”, i.e. tris-hydroxymethyl methane, either as the free base or as the carbonate
• a chelating agent such as EDTA, ethylene diamine tetracetic acid
• an anionic detergent such as SDS, sodium dodecyl sulphate
• uric acid or a urate salt optionally (d) uric acid or a urate salt.
• a particularly preferred solid medium according to this aspect of the invention comprises an absorbent cellulose-based paper such as filter paper having a minimal loading, per sq.cm. of paper, as follows: (a) EDTA: 0.5 micromols (146.1 mg of free acid) (b) Tris: 8 micromols (968.8 mg of free base)
• uric acid 2 micromols (336.24 mg of acid).
• uric acid or a urate salt in accordance with this invention has been found to be particularly desirable for the long term storage of DNA, as this component performs a number of functions. Firstly, it is converted into allantoin in acting as a "free-radical" trap that preferentially accepts free radicals, that would otherwise damage the base guanine in the DNA, (e.g. 2 - 3 ). Such free radicals are generated by the spontaneous oxidation of, for example, thio groups in the denatured serum protein, and may also be generated by the large amount of iron in blood 4 .
• the uric acid also acts as a component of the buffering system in that it is a weak acid. It also acts as an erodible surface in that it is sparingly soluble so that DNA- containing material dried onto its crystals will be released as the urate beneath them erodes.
• the composition may include a base, optionally a monovalent weak base, in an appropriate amount to cause an alkaline pH between 8.0 and 9.5 to be imposed upon the blood that is placed upon the matrix. This is to ensure the proper action of the chelating agent in binding divalent metals. It is also to prevent the action of acid nucleases that are not so dependent on divalent metals.
• the base may be a weak organic base, such as Tris.
• an inorganic base such as an alkali metal carbonate or bicarbonate, for example sodium, lithium or potassium carbonate or bicarbonate, may be used.
• the chelating agent is preferably a strong chelating agent such as EDTA, however a wide range of suitable strong chelating agents are commercially available.
• the function of the chelating agent is to bind divalent metal ions, magnesium and calcium, and also to bind transition metal ions, particularly iron. Both calcium and magnesium are known to promote DNA degradation by acting as co-factors for enzymes. Metal ions such as iron, that readily undergo oxidation and reduction, also damage nucleic acids by the production of free radicals 4 .
• the anionic surfactant or detergent is included in the composition of this aspect of the invention as the primary denaturing agent. Any strong anionic detergent that binds to and denatures proteins is suitable, and as well as SDS mentioned above other detergents such as sodium lauryl sarcosinate may also be used. This anionic detergent causes many pathogens to be inactivated due to the non-specific destruction of the secondary structure of their coat proteins, their internal proteins, and any membranes they may be dependent upon. There are exceptions, since the anionic detergent does not inactivate the most resistant bacterial spores, nor does it inactivate some extremely stable enteric virions. However, these exceptions are agents that are already likely to be transferred by ordinary contact and there is currently no great concern that these agents constitute a risk from blood.
• the cannula component of the present invention provides for sample processing utilizing an automatic fluid delivery system (AFDS) and a fluid reaction system associated with a solid material.
• AFDS automatic fluid delivery system
• a fluid reaction system associated with a solid material The problem hindering such use was due to obstruction of fluid flow into and out of the prior art cannulas or pipettes by the solid material.
• the cannulas or pipettes of the instant invention are advantageous over prior art pipettes used with AFDSs by permitting fluid flow into and out of the cannula even when the fluid reaction system is associated with a solid material.
• fluid includes both liquid and gaseous substances, as well as flowable suspensions.
• a “fluid reaction system” is any system wherein a starting material is transformed into the desired final product through the addition and/or removal of one or more fluid reagents.
• a fluid reaction system is conducted in a vessel of a selected size which may be varied according to the requirements of the fluid reaction system.
• the vessel size and composition may be of any size and material suitable for the process being performed.
• a suitable vessel composition is one which is non-reactive and impermeable to the reagents of the fluid reaction system.
• Fluid reaction systems amenable to utilization of the cannulas of the invention include, for example, PCR amplification, ELISA assay, oligoligase assay (OLA) and ligase chain reaction (LCR).
• the pipette or cannula is used to direct fluid into, or remove fluid from, the fluid reaction system.
• conventional pipettes were undesireable since they would become blocked by the solid material, when the pipette was used to "suck" fluid from the fluid reaction system.
• automatic fluid delivery system includes hand ⁇ held and robotic fluid delivery systems.
• automatic fluid delivery systems are devices which dispense and remove fluid reagents to and from individual wells of multi-well reaction plates.
• Hand-held automatic fluid delivery systems comprise a single plunger handle with multiple fluid aspirating and dispensing ends to simultaneously aspirate and dispense fluid of a fluid reaction system from single or multiple fluid reaction vessels simultaneously.
• Robotic automatic fluid delivery systems are computer operated rather than hand-held and include such products, for example, as BIOMEK 2000 (Beckman Instruments, Fullerton, CA) and Zymark Benchmate (Zymark, Hopkinton, MA).
• the cannula component is of a construction to inhibit obstruction of fluid flow into and out of the cannula even when the fluid reaction system is associated with a solid material.
• "associated” or “association with a solid material” means a solid particle which is present in the fluid reaction system such that it is capable of obstructing the aspirating or dispensing port of a cannula and hence inhibit proper functioning of the AFDS.
• a solid material associated with a fluid reaction may be present in the fluid reaction system to provide a chemical or mechanical function in the fluid reaction system.
• solid materials associated with fluids in a fluid reaction system which provide a chemical function include: solid material upon which a biological compound or biopolymer is adsorbed; a solid charcoal absorbent; C, derivatized silca; ion exchange resins; and/or polystyrene divinyl benzene.
• An example of a solid material associated with a fluid reaction system which can provide a mechanical function is a magnetic stir bar.
• the reference numeral 1 of Figure 2 depicts a pipette or cannula according to the present invention.
• a typical cannula 1 is a cylindrical tube.
• a useable cannula may, however, be of a geometric shape other than cylindrical.
• the cannula includes a shaft or wall 2 defining internal fluid flow channel 3 (F. 3).
• the cannula has a distal end 4 and a proximal end 5.
• the distal end 4 is that end which is inserted into the fluid of a fluid reaction system.
• the proximal end 5 is operatively connected to the AFDS, in use.
• the distal end 4 of the cannula wall 2 comprises at least two orifices.
• the orifices or "ports” are in fluid flow communication with the fluid channel 3.
• the ports are a primary fluid aspirating and dispensing port 6 ("primary port”) and at least one auxiliary fluid aspirating and dispensing port (“auxiliary port”) 7.
• the primary and auxiliary ports 6, 7 may be continuous or discontinuous. By “continuous” it is meant that there is no physical or mechanical separation between the primary port 6 and the auxiliary port 3. Additionally, the location of the auxiliary port 7 relative to the primary port 6 varies with the particular embodiment of the cannula. Both the primary port 6 and auxiliary port 7 contact the fluid of the fluid reaction system.
• the construction of the primary 6 and auxiliary 7 port of a cannula 1 according to the present invention is such that when a negative pressure is exerted by the AFDS, a single solid particle associated with a fluid of the fluid reaction system will not likely obstruct fluid flow into the distal end of the cannula 1, by simultaneously blocking both ports.
• the cannula 1 be constructed such that the primary port 6 and auxiliary ports 7 are positioned relative to one another in a preferred manner.
• the position of the ports 6, 7 should be such that at least a portion of one port will likely remain unobstructed by any solid material blocking the other port.
• the relative configurations and/or locations of the primary and auxiliary ports provide for this. For the embodiment shown in Figures 1 through 3, this is accomplished by positioning port 6 at an end of the cannula 1, and by positioning port 7 along a side of shaft 2.
• the AFDS is connected to a cannula.
• the exact mechanism by which the cannula connects to an AFDS will vary with the AFDS used.
• the connection for a cannula used with a ROSYS 3300, (ROSYS, Wilmington, DE) comprises .5 inch polypropylene tubing.
• Other methods of coupling include slip fittings and "screw on" connections.
• the coupler is shown at 8.
• the AFDS cyclicly exerts a positive and negative pressure through fluid channel 3. It is typically during the negative pressure phase that the fluid channel of the prior art pipette port opening becomes obstructed.
• the obstruction typically occurs as a result of the negative pressure drawing the solid material into the source of the negative pressure which, in prior art pipettes, is the pipette port opening in contact with the fluid of the fluid reaction system.
• a solid particle which is larger than the pipette port opening covers the pipette port opening and obstructs the negative pressure from effectively removing any remaining fluid.
• an edge of the cannula which may come into contact with the solid material is smooth, i.e. is relatively free of "burrs" or other imperfections which could cause adherence of the solid material to the cannula even in the absence of negative pressure.
• cannulas may be manufactured from materials commonly used to make fluid dispensing pipettes and tips which are known in the art.
• the material the cannula is composed of should preferably be chemically inert to conditions encountered when contacting a reagent of the fluid reaction system.
• Materials suitable according to the invention include malleable metals, for example, stainless steel, titanium, aluminum, platinum, gold, nickel and other inert metals.
• a cannula may be manufactured out of plastic resins or glass. For a plastic cannula to be used in a fluid reaction system containing phenol, inert, phenol-resistant resins are preferred.
• the dimensions for the disclosed cannula are for use with the above-listed robotic systems and the dimensions may be changed as needed for any specific system.
• the preferred length of the cannula is generally governed by the requirements of the AFDS used.
• the preferred diameter of the cannula is determined by the requirements of the AFDS and the diameter of the fluid reaction system vessel.
• the cannula should be of a size which can be operably inserted into the vessel of the fluid reaction system, if needed.
• the ports 6, 7 were discontinuous with one another and were spaced on cannula 1 such that they would not likely be simultaneously blocked by a single particle.
• the primary port 25 is an orifice at the distal most aspect 26 of the fluid channel 15.
• the opening of the primary port 25 is in a plane generally perpendicular to a longitudinal axis 27 of the fluid channel 15.
• the primary port diameter is approximately equal to an inside diameter of the fluid channel 15, although such is not required.
• the primary port inside diameter is about .010 to .1 mm, preferably .020 to .040 mm.
• the length of the cannula would be about 6 cm to 20 cm, preferably 15 cm.
• the term "about” refers to the quantitative dimensions stated and dimensions which are not necessarily quantitatively equal, but which are of a dimension which will effect a similar result.
• the cannula comprises at least one auxiliary port 24.
• the auxiliary port 24 is continuous with (i.e. is connected to) the primary port 25 and is in the shape of an inverted V.
• the inverted V has an apex 11 and a base 28.
• the apex 11 of the inverted V shape of the auxiliary port is up the cannula wall 19 from the base 28 in the direction away from the distal end 26.
• the width of the base 28 of the inverted V is about .010 to .030 mm, preferably about .020 mm and the height of the inverted V from the base 28 to the apex 11 is about 1.0 mm to 4.0 mm, preferably about 2.0 mm.
• One or more inverted V's may be placed circumferentially around the circumference of the cannula wall. When multiple inverted V's are used, the profile of the distal end of the cannula will have a "saw tooth" appearance. Therefore, according to this embodiment, a solid material in association with a fluid reaction system preferably does not likely simultaneously obstruct the primary 25 and auxiliary 24 port when a negative pressure is exerted.
• the primary port 25 and/or any additional auxiliary port 24 preferably remains unobstructed. If, on the other hand, the negative pressure causes a solid material to adhere to the primary port 25, preferably one or more auxiliary ports 24 remain unobstructed.
• FIG. 5 Another alternate embodiment of the invention is depicted on Figure 5.
• a cannula 35 comprising a primary port 36 at the distal end 37 of the fluid channel 38 is in a plane perpendicular to a longitudinal axis 39 of the fluid channel 38.
• the port 36 is then fitted with a porous frit 40 of about 0.20 ⁇ m to 0.60 ⁇ m porosity, preferably about 0.45 ⁇ m. Because of the porosity of frit 40 multiple pores are present. Hence, any single pore of the frit 40 may be considered an auxiliary port to the primary port, which would be other ports in the same frit 40.
• Frits suitable according to the invention can be manufacmred from such materials as spun glass, stainless steel, titanium and resins.
• a non-flat portion of the spherical frit 40 remains exterior to the distal most aspect wall 41.
• Application of a negative pressure, by the AFDS, to the fluid channel 38 in contact with a fluid reaction system containing a solid material will draw the solid against the frit 40.
• a solid material drawn against the frit 40 will adhere to the frit 40 tangentially and obstruct fluid flow at the tangential point of adherence.
• the remainder of the ports of the frit 40 will remain unobstructed thereby allowing fluid to flow into pores of the frit which are not blocked by the adhering solid material. It will be understood that alternate, non-flat, geometries for frit 40 could be used.
• the biopolymer purification apparatus component of the present invention provides an apparatus which removes salts and other low molecular weight compounds from biopolymers in a biopolymer reaction mixture. After contacting a biopolymer reaction mixture with the biopolymer purification apparatus, the purified polymer may, for example, be used in in vitro and in vivo biochemical reactions or be subject to analysis by, for example, capillary electrophoresis.
• biopolymer means any polymeric molecule utilized in biological systems derived from the same or different monomers (e.g., polypeptides, proteins, DNA, RNA, carbohydrates and similar biological molecules).
• the biopolymers may be chemically synthesized, genetically engineered or be derived from natural products.
• biopolymer reaction mixture is used herein to define any biopolymer in a solution in combination with salts or other low molecular weight compounds.
• by ⁇ products means compounds which are of a low molecular weight relative to a biopolymer including, for example, salts and/or residual reactants.
• Figures 7 and 8 show the biopolymer purification apparatus as preferably comprising a (rectangular) block 50 comprising a polymeric gel, preferably poly aery lamide. Within the block 50 are multiple wells or depressions 51 in the top surface 52. The wells 51 are formed into a shape which can hold a volume of fluid.
• the method for forming the wells 51 includes any method known for shaping polymeric gels (e.g., glass plate castings, injection molding and poured molds).
• the typical appearance of a biopolymer purification apparatus is similar to that of a multi-well plate.
• a biopolymer reaction mixture is placed into a well 51. After contact with the well for an amount of time, the low molecular weight by-products will diffuse from the well, as indicated by arrows 60.
• the biopolymer purification apparatus is flexible but dimensionally sufficiently stable for use with a robot controlled automatic fluid delivery system to locate and operate reliably on its micro- wells.
• a volume of biopolymer reaction mixture is placed into a well 51 of the biopolymer purification apparatus.
• the size of a typical well 51 of the biopolymer purification apparatus will be about 0.5 mm to 6.0 mm deep, preferably about 4.0 cm deep and about 0.25 cm to 2.0 cm in diameter, preferably about 0.3 cm to 1.0 cm in diameter.
• the amount of contact time between the biopolymer reaction mixture and the biopolymer purification apparatus will vary depending upon the application, but typically be about 5 to 40 minutes, preferably 10 to 30 minutes. What is required is a sufficient time for an acceptable level of purification to occur.
• the term "about” in this context refers to the quantity of time stated and times which are not necessarily quantitatively equal but of a duration which will yield a similar result. According to the invention, it is recognized that varying the concentration of the polymeric (polyacrylamide) components of the polymeric gel affects the amount of cross linkage between polymers. The amount of cross linkage directly affects the porosity of the gel.
• Polyacrylamide (a polymeric gel) is commonly used in gel electrophoresis to separate small biopolymer molecules.
• the biopolymer diffuses through the medium under the influence of an applied electric field.
• polyacrylamide gel is used to electrophoretically separate small DNA molecules by diffusion through the gel.
• the polyacrylamide gel is used as a medium for biopolymer purification without significant loss of the biopolymer by its diffusion into the gel.
• No applied electric field is used. Although no mechanism is asserted, one possible reason for the result discovered in the invention is that in an electric field, as applied in gel electrophoresis, a biopolymer such as DNA is highly orientated and can thus be drawn into the gel to move through it.
• biopolymer when such a biopolymer is not in an electric field, as in the present invention, it is randomly rotating and will have an effective molecular volume (or diameter) higher than it does in an applied electric field. Thus, the biopolymer is prevented from moving through the gel due to lack of an orientating electric field. Although the gel is a barrier to biopolymers, it is not a barrier to smaller molecules and ions, which can readily diffuse into the gel material leaving the biopolymer behind (in the vessel).
• the purified biopolymer solution can then be recovered from the vessel manually or by an automatic fluid delivery system.
• the biopolymer may be used in a subsequent biochemical processes or, for example, be analyzed. Production and use of the biopolymer purification apparams is described in Example 6.
• This aspect of the invention is a system for efficiently processing large quantities of biological samples. Referring to Figure 9, the system will be described with regard to the components (stations) comprising the system.
• the system 100 provides for identifying a sample 101, placing a sample into a reaction vessel for processing 103, processing a sample; and tracking the sample 102 through processing 103, permitting correlation of a sample with the corresponding process result. To be of greatest utility the system performs without cross-contamination or carryover between individual samples.
• the system 100 will be referred to as a Punch Technology System (PTS) or a "system".
• PTS Punch Technology System
• the system 100 is capable of identifying, tracking and processing any biological sample stored therein.
• Interactive networking of the identification 101, tracking 102 and processing 103 components of the invention is performed by the integral system data center (ISDC).
• the operations performed by the ISDC may be performed by a human being.
• the ISDC is a computer hardware and software system which provides interactive commands to the PTS.
• the term "identify”, “identification” and derivations thereof means the capacity to detect and store, or detect and send information about a sample to another component of the system, for correlation of process results with the appropriate sample.
• the term “tracking”, “track” and derivations thereof, means the capacity to follow and correlate a sample with its processing result.
• processing means any procedure whereby a sample is subjected to reaction, purification, buffering, washing or other steps to transform a starting sample into a desired final product.
• the PTS system has the ability to identify, process and track at least two and typically a large quantity of samples, quickly and efficiently.
• large quantity it is meant that the system can preferably identify, process and track at least two, preferably at least 2 to 2,000 samples. The ability to obtain results from a large quantity of samples quickly and efficiently makes the system 100 especially suited for large scale screening operations.
• Such screening has great utility in medical diagnosis and treatment, pathology, biochemical research, drug screening, design molecular biology, forensic science, genetic engineering and other areas where large sample screening is required. Specific areas of use include, for example, antigen antibody studies, histocompatibility, DNA fingerprinting and genetic diagnostics.
• the system will quickly and efficiently compare a single DNA sample, stored on a solid medium as described above, to a large database of DNA profiles also stored on a solid medium. See eg. "UK to set up DNA Database of criminals," 370 Nature 588 (Aug. 25, 1994).
• the system 100 provides for an automated system whereby a stored biological sample, for example blood derived DNA, can be identified, processed and tracked providing a final DNA product which is suitable for PCR amplification or restriction enzyme digestion.
• a typical method for storage of biological samples processed according to the invention is a biological sample desiccated on a solid medium such as cellulose, nitrocellulose, plastic, glass fiber, anion exchange paper, filter paper, other treated paper, cotton patches or other medium used in the art.
• a preferred storage media for DNA samples is described in Section I of the present invention.
• Figure 9 depicts a schematic of the punch technology system 100 of the present invention. Again, the system comprises the identification station 101, tracking station 102 and processing station 103.
• a sample to be identified, tracked and processed by the PTS is stored on a sample card 104.
• Numeral 106 indicates the "spots" of individual samples stored on the sample card 104.
• the sample card 104 is "marked” with indicia 105 for identification by the identification station 101.
• the sample cards 104 Prior to application of the system, the sample cards 104 are placed into a "stacker" 107 which orientates the stored cards 108 in such a way as to be accessible to the identification station 101.
• a barrier 109 is placed between individual stored sample cards 108 while in the stacker 107.
• Arrow 110 indicates a path sample card may take when moving into the indentification station 101.
• the identification station 101 recognizes the mark 106 of the selected card.
• the information obtained by the identification station is sent to the tracking station 102 for later correlation with results from the processing station 103.
• Arrow 111 depicts a path an identified sample card may take in moving to the tracking station 102.
• the tracking station 102 follows a selected sample 105 removed from a sample card 104 as it moves through the processing station 103.
• the tracking station 102 first locates a selected sample 105 on the sample card 104. Then the tracking station 102 functions to remove the selected sample 105 from the sample card 104.
• the selected sample 105 is received by a container in which the sample will be processed by the processing station 103.
• the tracking station 102 tracks the location of the selected sample 105 from the time the selected sample card arrives at the tracking station 102 until sample processing is completed.
• sample card 104 After a selected sample card is removed from a sample card 104, the sample card 104 preferably moves along a path depicted by arrow 112 to be collected at stacker 114. A barrier 116 is placed between sample cards in stacker 114, to prevent direct contact between collected sample cards in the stacker 114. The sample cards in the stacker 114 can be returned to stacker 107 or some other storage location.
• a sample 105 selected from a sample card may move along a path depicted by arrow 120 from the tracking station 102 to the processing station 103.
• the processing station 103 processes a selected sample to achieve the desired final product result.
• the result for the processing station 103 is then correlated with the selected sample 105 tracked by the tracking station 102 from the selected sample card.
• the processes conducted at the processing station 103 may be conducted directly on the selected cartridge without removing the sample from the card.
• the PTS provides for identifying, processing and tracking a biological sample which has been stored on a solid medium.
• a medium particularly suited for the system comprises a solid medium whereby processing of the sample does not require a step of initially removing the sample from its constituent medium.
• a suitable medium for use with a stored sample of DNA for example, is the solid matrix described in Section I of the present invention.
• a DNA sample first had to be removed from a storage medium before PCR amplification and/or restriction enzyme digestion could be performed. While a number of methods for removing DNA from a solid medium are currently in use, many of these methods require extensive sample manipulation including: boiling, multiple pipetting and centrifugation steps, desalting chromatography, precipitation, membrane dialysis and other methods known in the art.
• Utilization of the solid medium described in Section I of the present invention of the provides a stored sample suitable for PCR amplification and/or restriction enzyme digestion without the need for first removing the sample from the solid matrix.
• the matrix of Section I of the present invention permits automated processing of a DNA sample without the problems associated with other sample storage systems.
• a sample is stored on a solid medium or "card" 104.
• a card 104 may be of any size sufficient to hold a minimum of one drop of blood or other biological sample.
• a typical card would be of a rectangular shape. The dimensions of a typical card would be about 1 to 25 cm, preferably about 5 to 15 cm in length by about 1 to 20 cm, preferably about 5 to 10 cm. Cards of different geometrical shape and size may also be suitable in alternative applications of the invention.
• the solid medium or card 104 is preferably of a constituency for storage of the sample without alteration of the starting sample.
• a biological sample 105 for example DNA
• a biological sample 105 for example DNA
• the phrase "liquid medium” means a sample wherein the biological sample to be stored on the solid medium is suspended or solubilized. In general, the liquid will evaporate from the solid medium leaving the DNA or other suspended or solubilized molecule adsorbed to the solid medium.
• a single biological sample or multiple biological samples in a liquid medium is/are placed on the card in an arrangement suitable for sample location by a card locator mechanism (described supra).
• the card is marked 106, either before or after receiving the sample, for later recognition by the identifying mechanism (described in Section IV,A,Z).
• the liquid medium contained sample After the liquid medium contained sample has been placed on the card, the liquid medium evaporates and the card containing the desiccated sample is stored by suitable means known in the art, for example, file storage, polystyrene coated, - 25° C and other methods which preserve a sample on a solid medium.
• suitable means known in the art for example, file storage, polystyrene coated, - 25° C and other methods which preserve a sample on a solid medium.
• a preferred method for storage of DNA, for example, is described in Section I.
• the identification station of the PTS provides for the system to recognize and recall a sample card.
• Each card may contain at least one, or more, of the same or a different biological sample.
• a single card contains one or more of only a single biological sample.
• each sample card has a mark which is identified by the identification station of the system. The mark allows for recognition and recall of the biological sample makeup of a sample card. Recall of the biological sample makeup of the card may be made by the identifying mechanism at the time of identification. Alternatively, the identifying mechanism may send the identification information to the "card sample locator" (CSL) mechanism (described infra.) for recall of the biological sample makeup of a card.
• CSL card sample locator
• the identification station utilizes a "stacker" and an "identifying mechanism".
• the stacker provides a means for holding the cards prior to being identified by the identifying mechanism.
• a stacker may comprise a rack or cassette as used, for example in paper copying machines or other systems which hold paper or other "sheetlike" items.
• the stacker can take any of a variety of shapes provided it is of size and shape capable of holding the cards in an orientation permitting the identifying mechanism to identify a card. 11406 PCMB95/00989
• the cards are placed in the stacker in such a way as to prevent crossover contamination between cards or samples.
• Prevention of crossover contamination may be effected by placing a barrier between each card, preferably a non clinging barrier such as cellulose paper, wax paper, plastic and other barriers known in the art.
• the barrier is removed from between the cards before or after identification depending on the location of the identifying mechanism.
• the identifying mechanism of the system provides means for identifying the cards and the samples contained thereon.
• the card contains a "mark. "
• "mark” means a code or other means for the identifying mechanism to recognize the card.
• the cards may contain a bar code and the identifying mechanism is a bar code reader as used in grocery store check out lines, DNA repositories, bar code scanners on gas chromatograph/mass spectrometry autosamplers and high pressure liquid chromatography (HPLC) autosamplers.
• the cards may be identified by machine readable numbers or by other methods.
• the information identified by the identifying mechanism is then sent to the tracking station to track a sample on the sample card identified by the identifying mechanism for later correlation with the process results.
• a sample card may be identified by the identifying mechanism while in the stacker and subsequently moved to the tracking station. Alternately, a sample card may first be moved from the stacker across an identifying mechanism, identified and then moved to the tracking station.
• the tracking station of the system provides for tracking of an individual sample from a time after the sample card is identified by the identifying station until completion of the final step of the process being performed by the processing station.
• the ability of the system to track an individual sample through an entire process assures the accuracy of the system in correlating the proper sample with the appropriate result when processing a large quantity of samples.
• the sample tracking station has three main functions. It should first locate a sample on a card (possibly containing multiple samples). The tracking station then removes the located (selected) sample from the sample card for processing. (If the sample is removed for processing.) Finally, the tracking station maintains recall of the location of a selected sample as it moves through the processing station of the system.
• the three functions are termed the "card sample locator” (CSL) mechanism, the "punch mechanism” (PM) and the “receiving container sample locator” (RCSL) mechanism respectively.
• the CSL mechanism comprises a device capable of locating a spot on a sample card containing a single sample.
• information permitting the CSL mechanism to locate which sample on the card is to be processed comes from the ISDC.
• CSL mechanisms suitable for use according to the method of the invention include, for example, devices such as laser indexing systems, mechanical indexing systems, and optical density systems. Laser and mechanical indexing systems are used, for example, on photocopiers and facsimile machines.
• a punch mechanism comprises a blade, cutting laser, solid punch and other devices capable of cutting through the solid medium containing the sample.
• the removed sample is then placed into a receiving container.
• the size of the card sample which is removed from the card is obviously of a size which fits into the receiving container.
• a preferred embodiment of the punch mechamsm comprises a circular blade.
• the appearance of .J blade is similar to a skin biopsy punch or a cork bore.
• such blades are composed of stainless steel, titanium or other materials commonly used to make paper cutting blades.
• the diameter of the solid medium punched is about 2 mm to 10 mm, preferably about 4 mm in diameter.
• the surface of the blade which has come in contact with the card is preferably heat sterilized to miiiimize cross contamination of samples. Heat sterilization my accomplished using a propane flame or heating coil at a temperature of about 250 to 300°C, preferably about 275 °C.
• the sample removed by the punch mechanism is placed into a receiving container.
• receiving container includes a test tube, a single well of a microtiter plate, eppendorf mbe, PCR tube and other reaction container used routinely by those skilled in the art to process biological samples.
• the receiving container into which the removed sample is placed is detected by the "receiving container sample locator" (RCSL) mechanism.
• the RCSL may send the information of the location of a selected sample to the ISDC which may function to track the sample throughout the processing by denoting location and progress throughout the processing station.
• the card After a sample is removed from a sample card the card is restacked in a second stacker or cassette.
• the cards are placed in the stacker in such a way as to prevent crossover contamination between cards or samples. As described above, prevention of crossover contamination may be effected by placing a barrier between each card.
• the receiving container which contains a biological sample is then moved to the sample processing station.
• the sample processing station of the PTS provides for purification, buffering, washing, PCR amplification or other processes necessary to transform a sample into the processed final product. After a sample has been placed into a receiving container, the sample may be treated in situ. Alternatively, the sample to be processed may first be removed from the solid media. The process that the processing station applies varies with the type of sample and desired treatment goal.
• the processing station of the PTS may comprise an automated fluid delivery system with robotic mechanisms for moving the reaction container and for aspirating and delivering reaction fluids during sample processing.
• the use of automatic fluid delivery systems in the processing station is preferably performed utilizing the cannula system disclosed in Section II of this invention.
• the processing station of the PTS may first subject a sample to a process for removing the sample from the solid medium (i.e. punched sample card). In this situation, the steps of the process would involve treating the sample with the appropriate reactants needed to desorb the stored sample from the solid medium.
• the sequential steps for desorbing a sample from its storage medium are known in the art.
• the sample may remain adsorbed to the solid medium throughout the entire reaction process.
• DNA adsorbed to a solid medium may ultimately be amplified by PCR or digested by restriction enzymes in the same receiving container.
• the DNA is preferably rendered free of proteins and other impurities contained in the adsorbed sample.
• the processing station may render the DNA free of protein and other impurities using the protocol described in Example 3 and 4.
• the processing station is preferably equipped with four independently controlled heat/cold zones. Of these, one zone is preferably at about 4°C, a second zone is preferably at 54°C, a third zone is preferably at about 96°C and a fourth zone is preferably at about 37 °C.
• cross contamination means contamination of one sample by another.
• cross contamination means contamination of one reagent by another.
• the pipette tip used for aspirating and dispensing fluids during PCR amplification are preferably used for only a single reagent and only a single sample.
• Such prevention of cross contamination may be accomplished using automatic fluid delivery systems.
• pipette tips analogous to automatic fluid delivery system cannulas described earlier in the invention may be produced for single application use. According to this aspect of the invention, after a single use of a pipette tip, the tip is disconnected from the automatic fluid delivery system.
• a new pipette tip is then placed on the automatic fluid delivery system before the AFDS moves to the next sample or reagent. Once installed, the new pipette tip is used to dispense a single fluid and the process for pipette tip replacement is repeated.
• EXAMPLE 1 Collection and Extraction of DNA: Sodium dodecyl sulphate was applied to Whatman 3mm paper in a solution such that there was approximately 50 ⁇ l per sq.cm. of a solution of 2% sodium dodecyl sulphate, lOmM EDTA, and 60mM tris (free base), i.e. approximately 1 mg of sodium dodecyl sulphate per sq.cm. The paper was then dried.
• the treated paper was soaked with drops of blood from various primates.
• the blood-stained paper was dried, sent through the ordinary mail so that it spent at least three days in the mail, and then had the DNA extracted from it using standard procedures involving detergent-aided proteolysis and phenol extraction of the paper.
• the resultant DNA was then tested for its quality by being digested with restriction endonucleases and the fragments analyzed by agarose gel electrophoresis.
• the DNA fragments were found to be as high in quality as DNA produced from fresh blood. This demonstrates that the DNA can be extracted from detergent-treated papers and that the DNA is of sufficient quality for most normal purposes.
• Record cards can be prepared in batches and stored until needed. Whatman No.1 paper about 10cm x 15cm in size and with appropriate places marked out with an "indian ink” (i.e. colloidal carbon ink-stamp) is suitable, and any special notes on the cards can be made with an ordinary "lead” pencil (i.e. Graphite pencil).
• the cards are marked out in a regular pattern to assist in systematic storage and retrieval of DNA samples. Marked cards are wrapped in clean paper, then foil, and autoclaved with a dry cycle. They are then treated with a solution of 40mM uric acid and 100mm tris (free base). The function of the urate is to protect the DNA from aging and to aid the desorption from the paper if required. These treated record cards can then be kept until required.
• DNA to be stored is taken up in a dilute alkaline buffer containing EDTA, e.g. TE buffer (Tris-EDTA pH 8.0).
• TE buffer Tris-EDTA pH 8.0
• approx 1 ml of bacterial culture containing plasmid is treated by the alkaline lysis method, with one phenol extraction and one alcohol precipitation, to get approx 50 ⁇ l of plasmid or other DNA in TE buffer.
• a 5 ⁇ l aliquot of each DNA sample is used to make a spot on the urate treated record card.
• the cards are conveniently stored in a sealed container in a refrigerator freezer (about -15°C) in the presence of drying agent such as silica gel and a few grains of dry sodium carbonate to remove any traces of acid vapours.
• drying agent such as silica gel and a few grains of dry sodium carbonate to remove any traces of acid vapours.
• the desorption of DNA samples, both single and double stranded, from Whatman No.1 paper soaked with a solution of 40mM uric acid, and lOOmM tris (free base) was examined by using the plasmid pUC19 as a source of standard double stranded DNA and M13 as a source of single stranded DNA.
• EXAMPLE 3 In situ Use of Stored Blood DNA in PCR.
• Blood DNA stored on filter paper treated in accordance with the present invention can be amplified in situ by the polymerase chain reaction (PCR) technique.
• the treated paper used in this Example was Whatman 3 mm paper treated with a solution comprising, per sq.cm of paper, 2 micromols uric acid, 8 micromols tris (free base), 0.5 micromols EDTA and 1 mg SDS.
• the stored blood DNA was treated to remove protein, then washed to remove phenol and add suitable ions, prior to DNA amplification.
• Phenol solution A suitable mixture is phenol, 50 gm containing 120 mg of 8-hydroxyquinoline that has been saturated with 10 ml of 1.0 M tris-acetate pH 8.0 and 1.0 ml 2-mercaptoethanol. After saturation by shaking at room temperature, the aqueous phase is thoroughly removed and discarded.
• Solution B A suitable mixture is phenol, 50 gm containing 120 mg of 8-hydroxyquinoline that has been saturated with 10 ml of 1.0 M tris-acetate pH 8.0 and 1.0 ml 2-mercaptoethanol. After saturation by shaking at room temperature, the aqueous phase is thoroughly removed and discarded.
• Solution B A suitable mixture is phenol, 50 gm containing 120 mg of 8-hydroxyquinoline that has been saturated with 10 ml of 1.0 M tris-acetate pH 8.0 and 1.0 ml 2-mercaptoethanol. After saturation by shaking at room temperature, the aqueous phase is thoroughly removed and discarded.
• Solution B A suitable mixture is phenol, 50 gm
• All steps are preferably carried out in a single tube made of a suitable phenol resistant material, e.g. polyethylene.
• step (b) Removal of phenol and addition of suitable ions: the paper in its tube from step (a) above, is rapidly washed in three lots of 1 ml of solution B. Washes are at room temperature and are simple additions followed by aspiration to waste. The paper is then washed for 20 minutes at room temperature with solution C. (This is to saturate the DNA on the paper with Magnesium ions and remove the last of the phenol.) The solution C is aspirated to waste and the paper is solvent-dried with one wash of pure isopropanol and then vacuum dried.
• the final DNA-paper should be quite white without any obvious remnants of the red-brown colour of blood. It is now ready for use in a PCR reaction mix.
• the treated DNA-paper as described above has been shown to be a suitable substrate for DNA polymerase chain reaction (PCR) amplification of DNA.
• PCR DNA polymerase chain reaction
• Extracted DNA DNA from 10 ml of blood obtained from a male volunteer was extracted by standard protocols.
• Treated DNA Filter Paper Blood specimens from the same volunteer were applied directly to treated filter paper with subsequent treatment as described above. The paper was cut into about 1mm 2 pieces for use in
• Target No. 1 Region of exon 2 of the n-Ras proto-oncogene on chromosome 1.
• the primers used are:
• Rl 5' TGACTGAGTACAAACTGGTGGTG3' and R2: 5' CTCTAT GGT GGGATCATATTC A 3'.
• the amplified DNA fragment obtained with these primers is HObp in size.
• Target No. 2 A male specific Y chromosome repeat sequence.
• the primers are:
• 007 5' TGGGCT GGA ATG GAA AGGAATGCA AAC 3' and 008: 5' TCCATTCGATTCCATTTTTTTCGAGAA3'.
• the amplified DNA fragment obtained with these primers is 124 bp in size.
• Target No.3 A male specific Y chromosome repeat sequence.
• the primers used are:
• 004 5' GAATGT ATT AGAATGTAATGAACTTTA 3' and 006: 5' TTCCATTCCATTCCATTCCTTTCCTTT 3'.
• the amplified DNA fragment obtained with these primers is 250 bp in size.
• Extracted DNA (1 ⁇ g) or about 1mm 2 fragments of treated DNA filter paper were placed into 0.5ml Eppendorf tubes and made to 25 ⁇ l in
• PCR reaction mixture consisting of: 67 mM Tris HC1 (pH 8.8 @ 25 °C) 16.6 mM ammonium "Ifate 2 mM MgCl 2 0.01 % (w/v) gelatii
• Lanes 1-3 target No. l , lane 1 : 1 ⁇ g DNA, lane 2: 1mm 2 filter, lane 3: no DNA control;
• Blood proteins were extracted from the treated paper by a procedure using Solution A as in Example 3. Blood proteins were also extracted from samples using two procedures involving a modified Solution A (Solution A4 and Solution A7). Extractions were carried out on samples made with a 0.5 x 0.5 mm leak punch (B.Y.T. Co., Baycity, MI) and placed in 1.7 ⁇ l Eppendorf tubes. 11406 PCMB95/00989
• the protein-free adsorbed DNA was then amplified using PCR methodology.
• Procedure 1 Add 500 ⁇ l to each mbe Solution A (50ml of Ultra pure liqu Phenol, 10ml IM TE buffer, 120mg 8-Hydroxyquinoline, 1ml 2-0 Mercaptoethanol. Allow to sit 15 minutes to let aqueous and organic layer to separate. Remove aqueous layer. Incubate 1.5 hours at 55°C. Wash IX with Solution A. Wash 3X with Solution B (75% v/v Isopropanol, 25% v/v Potassium Acetate at pH 7.8). Incubate at room temperature for 20 minutes with Solution C (75% v/v Isopropanol, 25% v/v Magnesium Acetate). Solvent dry paper with pure Isopropanol then Vacuum dry 10 minutes. Paper should be white and ready for PCR.
• Procedure 2 Add 500 ⁇ l to each tube Solution A4 (50ml Phenol/chloroform/Isoamyl, 10 ml IM TE buffer, 120 mg 8- Hydroxyquinoline, no 2-/3 mercaptoethanol was added. Allow to sit 15 minutes, remove top layer). Incubate 1.5 hours at 55 °C. Wash IX with Solution A4. Wa ⁇ 3X with Solution B (75% v/v Isopropanol, 25% v/v Potassium Acetate at pH 7.8). Incubate at room temperature for 20 minut vith Solution C (75% v/v Isopropanol, 25% v/v Magnesium Acetate).
• Procedure 3 Add 500 ⁇ l to each mbe Solution A7 (a suitable digestion buffer such as lOmM Tris, 5 mM EDTA, 0.5%SDS at pH 7.8 plus 3 ⁇ l Proteinase K). Incubate at 55°C for 1 hour. Deactivate enzyme at 95°C for 10 minutes. Incubate 1.5 hours at 55 °C. Wash IX with Solution A7. Wash 3X with Solution 1406 PCMB95/00989
• Amplitvpe * HLA DO ⁇ (Perkin Elmer, Foster City, CA) 25 ⁇ l Master Mix + 25 ⁇ l 8 mM MgCl 2 + paper overlay with 40 ⁇ l Mineral Oil in .5 ⁇ l Gene Amp Thin- walled tubes. Thermocycler parameters are: 94°C/30 sec, 94°C/10 sec, 60°C/10 sec, 72°C/10 sec (32X), 72°C/10 min, followed by 4°C soak.
• Amplitvpe '* PM (Perkin Elmer, Foster City, CA) 40 ⁇ l Master Mix + 40 ⁇ l 6mM MgC 1 2 + paper in Micro- Amp tubes. Thermocycler parameters are: 95°C/1 min, 95°C/30 sec, 63°C/30 sec, 72°C/30 sec (32X), 72°C/10 min, followed by 4°C soak. DQ ⁇ and Amplitype TM PM were typed using protocol outlined by Perkin Elmer.
• FIG. 2 This example is for preparation of a preferred embodiment of a cannula according to the invention as depicted on Figures 2 and 3.
• a standard cannula for use with a ROSYS 3300 (ROSYS, Wilmington, Delaware) was obtained from ROSYS (Wilmington, Delaware).
• the length of the cannula is 15 cm.
• the inside diameter of the primary port 6 is .040 mm.
• auxiliary ports 7 of .020 mm in diameter were drilled at about 180° apart from each other using a titanium drill bit.
• the edges of the primary and axillary port 6, 7 were smoothed using a file.
• the completed cannula was coupled to a ROSYS 3300 (ROSYS, Wilmington, Delaware) automatic fluid delivery system. Once connected, the automatic fluid delivery system with the cannula disclosed according to the invention was put to use for aspirating and dispensing fluids from a fluid reaction system associated with a solid.
• the solid was a small sample of me solid storage medium containing DNA as described in Example 1.
• This example is for preparation of a standard poly-acrylamide gel to be used to form a biopolymer purification apparams.
• Other hydrophilic polymers having similar properties can be used.
• the polymer was prepared as follows: 12 ml water 2 g acrylamide 40 mg N , N ' -methylene-bis-acrylamide
• the polymer reaction mixture was cast into rectangular blocks of a convenient size (e.g., 8.0 mm deep by 50 mm long and 30 mm wide) with regularly spaced small raised dimples (which create the micro-wells) in its bottom surface.
• the individual wells were about 4.0 mm deep and 4.0 mm in diameter.
• the poly-acrylamide is allowed to set in the mould about 30 minutes and then placed in distilled water overnight.
• the final product is approximately 14% water soluble plastic, but after desiccation the apparams is about 12.2% dry weight of polymer.
• the apparatus may be sealed in plastic bags made from polyethylene, for example.
• the material of the apparams has a very long shelf life stored with minimal care but preferably 25 °C or below and in the dark.
• DNA reaction mixtures from DNA amplification by PCR are placed into separate micro- wells of the apparams at room temperature, either manually or automatically by a cannula under robotic control. After sufficient time for the level of contamination to be reduced (e.g., 2 to
• the solutions of DNA in the micro- ells can be sampled either manually or by a robotic cannula and the DNA isolated or subjected to analysis by, for example, capillary electrophoresis.
• the material of the apparams can be cleaned for re-use by washing with distilled water overnight, however, the material is relatively cheap to produce and normally is discarded after use to avoid any possibility of contamination of subsequent samples.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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