AU2007200449B2

AU2007200449B2 - Injection port

Info

Publication number: AU2007200449B2
Application number: AU2007200449A
Authority: AU
Inventors: John Corbett
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2007-02-02
Filing date: 2007-02-02
Publication date: 2009-07-23
Anticipated expiration: 2027-02-02
Also published as: AU2007200449A1

Description

- 1 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant/s: Corbett Life Science Pty Ltd Actual Inventor/s: John Corbett Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 n SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: INJECTION PORT The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 52913AUP00 -2 FIELD OF THE INVENTION The present invention relates to a continuous flow system and in particular to a port for introducing a volume of a liquid sample into a continuous flow system. However, it will be appreciated that the invention is not limited to this particular field of 5 use. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field. 10 The prior art continuous flow apparatus are typically operated under positive pressure and require pumps for pumping a carrier fluid through a continuous tube/conduit such as a reaction tube. Typically, these pumps are high or very high pressure pumps. Accordingly, these prior art continuous flow apparatus require specialised high-pressure injection ports for delivering a liquid sample into the stream of 15 carrier fluid being pumped under high pressure through the tube. These high-pressure injection ports suffer a variety of drawbacks. However, the major drawback is their propensity for cross contamination between samples such as for example contamination of samples during loading, largely due to the septum-needle arrangement for injecting the sample, or between samples as they travel down the tubing. 20 Thus there still remains a need for improved continuous flow systems as well as sample handling and delivery means. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the abovementioned prior art, or to provide a useful alternative. SUMMARY OF THE INVENTION 25 The invention provides a sample port for use with continuous flow systems, particularly those in which samples are drawn through a column or a tube rather than pumped through under pressure. According to a first aspect the present invention provides a port for introducing a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow 30 tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said port comprising: a reservoir for continuously supplying said inlet with said fluid carrier, said reservoir adapted to maintain a substantially constant level of fluid carrier above said inlet and being fluidly engageable with said inlet of said continuous flow tube such that, in use, said fluid carrier stream and said P:WPDOCSGRS\Cobt\20417486 d-1.IoW2009 -3 liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure and when said fluid carrier is chosen such that its properties are sufficient to maintain the physical shape of the liquid sample introduced therein. 5 In an embodiment the liquid sample is an aqueous sample and the fluid carrier is a hydrophobic liquid. The fluid carrier can be suitably an oil such as a silicon oil. A sample port according to claim 3, wherein the hydrophobic liquid is an oil. According to a second aspect the present invention provides a continuous flow apparatus comprising the sample port according to the first aspect. 10 In an embodiment the continuous flow apparatus is a thermocycling apparatus for performing nucleic acid amplification reactions such as, for example, the apparatus described in PCT/AU02/0l112 (publication no. WO 2003/016558). One method of amplifying the nucleic acids is PCR. According to a third aspect the present invention provides a method for introducing 15 a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said method comprising the steps of: providing a port according to the first aspect; fluidly engaging said inlet of said continuous flow tube with said reservoir; introducing said fluid carrier into said reservoir and 20 introducing said liquid sample into said fluid carrier, said fluid carrier chosen such that its properties are sufficient to maintain the physical shape of the liquid sample and such that said fluid carrier stream and said liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure. According to a fourth aspect the present invention provides a method for 25 introducing a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said method comprising the steps of: providing a port according to the first aspect; fluidly engaging said inlet of said continuous flow tube with said reservoir; introducing said fluid carrier into said reservoir; immersing a 30 liquid sample dispenser into said fluid carrier contained in said reservoir; dispensing said P:WPDOCS\GRS\Corbd\2047486 doc./06/2009 -4 liquid sample adjacent said inlet and optionally manoeuvring said dispensed liquid sample with said liquid sample dispenser such that said dispensed liquid sample is introduced into said inlet and drawn through said continuous flow tube, said fluid carrier chosen such that its properties are sufficient to maintain the physical shape of the liquid sample and such 5 that said fluid carrier stream and said liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure. Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the 10 sense of "including, but not limited to". BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a side view of apparatus according to a first embodiment of the present 15 invention, shown prior to introducing a liquid sample into the continuous flow tube inlet; Figure 2 is a view similar to Figure 1 but showing the liquid sample introduced into the reservoir of fluid carrier; Figure 3 is a view similar to Figure 2 but showing the liquid sample drawn into the continuous flow tube inlet; and 20 Figure 4 is a side view of an alternative apparatus. DEFINITIONS In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the 25 invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as cQmmonly understood by one having ordinary skill in the art to which the invention pertains.

P:WPDOCSZRS\Corbe\20417486 doc-I/06/200 - 4a The terms "drawn" and "drawing" in reference to the fluid carrier stream being drawn through the continuous flow tube when a suction force is applied to the continuous flow tube outlet are to be distinguished from "pushing", "propelling" or "pumping" the fluid carrier stream through the continuous flow tube when a pumping force is applied to 5 the inlet. A pumping force is typically provided by the use of a high- pressure pump, such as a HPCL pump. In contrast, a suction force is typically applied by the use of a suction/aspirating/vacuum pump, optionally in conjunction with a vacuum bottle-type arrangement. For the purposes of the present invention a pumping force may be considered to be a positive force and a suction force a negative force.

-5 Furthermore, for the purposes of the present invention the reservoir is considered to be free from an applied pressure substantially greater than atmospheric pressure when the carrier fluid is being drawn through the continuous flow tube. A fluid may also be "drawn" through the continuous flow tube by the effects of gravity (when certain carrier 5 fluids are used). The term "atmospheric pressure" as used herein is intended to refer to an atmospheric pressure at substantially 10 1.325 kPa (i.e. 760mm Hg) or its equivalent at different altitudes. However, the skilled person will appreciate that this term permits a degree of variation and that the number is not to be construed as a precise value. 10 DESCRIPTION OF THE PREFERRED EMBODIMENT As will be appreciated, the port according to the present invention allows the introduction of liquid samples into a continuous flow column without the need for prior art high-pressure injection ports or specialised injection apparatus. The port is relatively inexpensive compared to prior art high-pressure injection ports, has no moving parts and 15 has no components that wear out (such as septa). Further, the port according to the present invention allows sample loading at atmospheric pressure with standard air displacement pipette tips which, since they are relatively inexpensive compared to needle-syringe injection apparatus, can be easily batch-sterilised and each tip discarded once used, thereby eliminating sample cross-contamination. In contrast, prior art needle 20 syringe injection apparatus must be cleaned/sterilised between sample injections. Further still, the "touch off' loading technique is "zero contact", meaning there is no contamination within the loading port. Yet further still, the port according to the present invention is particularly suitable for automatic sample loading by virtually any commercially available laboratory robotic system. 25 Preferably the fluid carrier is "drawn" (as opposed to "pushing" by pumping under high pressure) through the continuous flow tube by applying a suction force to the tube outlet. Drawing the fluid carrier stream through the continuous flow tube provides cost benefits compared to prior art devices since there is now no need for high-pressure pumps and, more importantly, no need for high pressure injection ports. However, in an 30 alternative embodiment the fluid carrier is chosen such that the fluid carrier will be drawn through the continuous flow tube under the effects of gravity. The suction force applied to the outlet of the continuous flow tube may be relatively easily provided by, for example, engaging the outlet of the continuous flow tube to a simple vacuum pump arrangement. For example, applying a vacuum of about -6 10 to 100 kPa to a 15-metre length continuous flow tube having a 1 mm internal diameter provides a flow of between about 50 to 500 pL/min. However, it will be appreciated that the flow rate is proportional to the internal diameter of the tube and/or the grade of oil and/or the amount of vacuum applied to the tube outlet. The tube could 5 even be gravity fed by appropriate choice of grade of oil and tubing internal diameter. The skilled person will appreciate that other vacuums and flow rates can be provided according to the particular application. Preferably the vacuum should be controlled to maintain an even flow rate through the continuous flow tube. Preferably the reservoir is open to, or held at atmospheric pressure, thereby 10 providing a "zero pressure loading port". The preferred reservoir includes a centrally tapered base which is adapted to captively receive a continuous flow tube. However, in an alternative embodiment the reservoir may be pre-fitted with a length of conduit such that the outlet of the pre-fitted length of conduit may be fluidly engaged with the inlet of a pre-existing continuous flow tube. To ensure that no air is entrained in the continuous 15 flow tube, the reservoir is configured such that the tube inlet is submerged within the volume of fluid carrier contained in the reservoir when the reservoir is fully charged with fluid carrier. The tube inlet is preferably substantially vertically configured to receive a liquid sample from above. Preferably a portion of the continuous flow tube intrudes into the reservoir such that the continuous flow tube inlet is disposed at about 20 the centre of the hight of the volume of fluid carrier contained in the reservoir when the reservoir is fully charged with fluid carrier. A suitable pump, such as a peristaltic pump, supplies the reservoir with fluid carrier and an optical sensor maintains pre-determined fluid level by controlling the rate of addition of fluid carrier into the reservoir. Alternatively, a weir-type arrangement may be provided for maintaining the fluid level. 25 However, other arrangements will be apparent to the person skilled in the art providing that the reservoir is sufficiently maintained with fluid carrier at substantially atmospheric pressure. A liquid sample may be introduced into the continuous flow tube by firstly positioning the tip of a sample dispenser, such as the tip of a pipette, just above the 30 continuous flow tube inlet, and slowly dispensing the liquid sample. Preferably the liquid sample is about 20 tL. However, the liquid sample may be as small as 1 PL or as large as 50 pL. Due to surface tension effects the aqueous liquid sample dispensed into the hydrophobic oil carrier is substantially spherical in shape. Optionally the tip of the -7 pipette remains in contact with the sphere of liquid sample to assist in manoeuvring the sphere to the continuous flow tube inlet and prevent it from "falling away" to the wall of the reservoir. Since there is a flow of fluid carrier into the continuous flow tube inlet the sphere of liquid sample can then be "touched off' into the inlet from where it will be 5 drawn into the continuous flow tube by the suction force applied to the outlet. It will be appreciated that a plurality of liquid samples may be introduced into the continuous flow tube at timed intervals. The liquid sample may be the same or different samples. Preferably, although not necessarily required, an intervening "wash" fluid is added between samples. To explain, a preferred sample loading comprises the following 10 sequence: wash-sample-wash-sample-wash- etc. Of course each wash/sample fluid are separated by carrier fluid. This sequence reduced sample cross-contamination since any portion of the sample which may become dislodged is "caught" by the wash fluid and not by the preceding sample. Preferably the wash fluid is water. The sample port of the present invention is particularly suitable for high 15 throughput automated systems. In an embodiment of the present invention the sample port may be charged with a plurality of sequential samples (and wash fluid doses if required) by way of a robotic sample handling system. In other embodiments of the sample port for high throughput applications, a plurality of continuous flow tubes may be provided. The continuous flow tube inlets are preferably spaced into an array within 20 the reservoir. This embodiment is also conducive for robotic sample handling whereby a robotic system can introduce a plurality of liquid samples into the plurality of continuous flow tube inlets. Alternatively, the array of continuous flow tubes may merge downstream in a parallel configuration into a single continuous flow tube thereby defining a "parallel" manifold. In this embodiment the liquid samples are loaded 25 sequentially from one end of the manifold to the other thereby allowing the liquid samples to be evenly spaced as they are drawn into and travel through the continuous flow tube. However, in other embodiments the array of continuous flow tube openings may merge downstream into a single continuous flow tube in series thereby defining a "series" manifold. Advantageously, in this embodiment the liquid samples may be 30 loaded simultaneously. In an alternative aspect, a low-pressure sample loading port is provided for use in a high-pressure continuous flow system. In this aspect, a pair of spaced tubes are provided which are housed between a pair of spaced rotatable plates to define a rotatable stage. In one position, one of the tubes is open to the atmosphere whilst the other is in- -8 line with the high-pressure continuous flow tube. The tube that is open to the atmosphere contains fluid carrier and may receive a liquid sample. Once a liquid sample is loaded the stage is rotated such that the tube open to the atmosphere is switched "in line" leaving the other tube available to receive a subsequent liquid sample. The process 5 is then repeatable. It will be appreciated that this port arrangement provides sample introduction via disposable tip and no needle-syringe apparatus is required since there is no pierecable septum and hence reduced possibility of sample cross contamination. The sample port according to the present invention is easily adaptable to many types of continuous flow apparatus. Apart from the port itself, the only substantive 10 change to these existing apparatus is the provision of a suction force to the outlet of the continuous flow tube, if gravity feeding is insufficient, to draw the fluids therethrough rather than "pushing" the liquid stream through via high-pressure pumping. The suction force, where required, can be supplied by a standard vacuum pump or the like. In one embodiment, the present invention is suitable for any continuous flow 15 device operated with a suction force. For example, it may be adapted for use with the continuous flow devices described in PCT Publication No. WO 03/016558. According to WO 03/016558, a fluid carrier stream interrupted by a plurality of liquid samples is pumped under pressure through a continuous flow tube coiled about a cylindrical heat exchanger having a plurality of different temperature zones. The temperature zones are 20 chosen to provide denaturation of nucleic acid into its component strands; annealing of oligonucleotide primers to complementary sequences in the nucleic acid; and synthesis of new nucleic acid strands. The liquid samples flowing though the continuous flow tube are subject to these varying temperatures in a cyclical fashion until a desired level of amplification has been achieved (amplification scaling with the number of times the 25 continuous flow tube is coiled about the heat exchanger). These and similar prior art devices necessarily require the use of high pressure pumps to force the fluid carrier stream through the continuous flow tube and a high pressure injection port to introduce the liquid samples into the fluid carrier stream. This equipment is relatively complex, expensive, requires regular maintenance and skilled operators. In contrast, the present 30 invention advantageously avoids the use of such high-pressure equipment by employing the novel port as described herein and drawing rather than pushing/propelling/pumping the fluid carrier stream through the continuous flow tube. The fluid carrier utilised is preferably devoid of biological contaminants (e.g. extraneous nucleic acids such as RNA or DNA), particularly when conducting PCR -9 using a continuous flow system, and is chosen to substantially prevent contamination between the liquid samples flowing through the continuous flow tube. However, in the present invention the fluid carrier is also preferably chosen to maintain the physical properties of the liquid sample. The applicant has found that silicone oils are 5 particularly suitable for the present invention. Ideally the fluid carrier is a silicone oil having a viscosity of between about 5 to 50 centistokes. However, it will be appreciated that the oil viscosity is not limited to this range. Without wishing to be bound by theory it is believed that suitability of silicon oils is primarily due to the relatively uniform chain lengths. Thus, any oil of suitable viscosity and having these chain-length 10 characteristics would be useful as a fluid carrier. The applicant has also observed that preferred silicone oils are those which provide a neutral buoyancy to the liquid sample, i.e. those having a density between about 0.95 to 0.99 g/cc. However, it will be appreciated that the oil density is not limited to this range. Preferably the liquid sample is a mixture of components for a PCR experiment. 15 For example, the liquid sample includes a sample to be analysed or reacted and reagents to be used in the analysis or reaction. Preferably the sample to be analysed or reacted is a nucleic acid such as DNA or RNA containing sample. Other components of the sample will typically include oligonucleotide primers, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), 20 deoxythymidine triphosphate (dTTP), and at least one of a thermostable DNA polymerase, enzymatically active fragments thereof, an enzymatically active derivative thereof and a reverse transcriptase. The continuous flow tube is preferably substantially transparent, resilient and formed from an inert material having a hydrophobic internal surface. For example, a 25 continuous flow tube formed from Teflon or Tefzel or similar material is preferred. However the continuous flow tube can be formed of any suitable material which will allow fluids to be drawn therethrough under a suction force. Preferably the internal diameter of the tube is between about I to 1.6 mm. It will be appreciated that whilst the sample port of the present invention will be 30 particularly suitable for a continuous flow PCR apparatus, the apparatus and method of the present invention is not limited to this field. For example, the present invention will be suitable for protein disassociation systems and isothermal reactions wherein the sample fluorescence is measured every cycle. However, the present invention is - 10 particularly suitable for robotic methods for reduced contamination loading of a sample into continuous flow systems. Reference will now be made to the drawings wherein like reference numerals refer to like parts throughout. Turning initially to Figure 1, the present invention 5 provides apparatus in the form of a port I and a method for introducing a volume of a liquid sample 2 into a fluid carrier 3 stream flowing into and through a continuous flow tube 4. The continuous flow tube 4 is preferably formed from Teflon and comprises an outlet (not shown) and a common inlet 5 into which the fluid carrier 3 stream and the liquid sample 2 are both introduced. The port 1 comprises a reservoir 6 for continuously 10 supplying the inlet 5 with the fluid carrier 3. Since the reservoir 6 is preferably configured to be open to the atmosphere the fluid carrier 3 stream may be drawn through the continuous flow tube 4 when sufficient suction force is applied to the outlet. As discussed above, the present invention is particularly suitable for a continuous flow device for the amplification of nucleic acids using the PCR. Typically, in these 15 devices a plurality of liquid samples 2 are introduced into the continuous flow tube 4 wherein each of the liquid samples 2 are separated by a volume of fluid carrier 3 to prevent contamination between liquid samples 2. In prior art devices this stream of liquid samples 2 and fluid carrier 3 is pumped through the continuous flow tube 4 and the continuous flow tube 4 is exposed to at least one temperature zone provided by a 20 suitable heat exchanger 7. However, in the present invention the stream of liquid samples 2 and fluid carrier 3 is drawn through the continuous flow tube 4 by applying a suction force to the outlet of the continuous flow tube by, for example, a vacuum-bottle type arrangement 8. The liquid sample 2 is a mixture of liquids for a PCR experiment and the fluid carrier 3 is an oil which is preferably devoid of extraneous biological 25 contaminants, e.g. nucleic acids such as RNA or DNA. Referring to Figure 1 again, the reservoir 6 preferably includes a centrally tapered base 9. The continuous flow tube inlet 5 is submerged within the volume of fluid carrier 3 contained in the reservoir 6 and is vertically configured to receive a liquid sample 2 from above. A weir 10 is provided for maintaining the level of fluid carrier 3. 30 The surface 11 of the reservoir 6 is open to atmospheric pressure and a pump (not shown), such as a peristaltic pump, supplies the reservoir 6 with fluid carrier 3 through inlet 12. The over-flow 13 of fluid carrier 3 in well 14 is returned to the pump for recycling.

- II Referring now to Figures 1 to 3, the method of the invention comprises firstly engaging a continuous flow tube 4 to the reservoir 6 containing the fluid carrier 3 and applying a suction force to the outlet such that the fluid carrier 3 is evenly drawn through the continuous flow tube 4. A liquid sample 2 is then introduced into the continuous 5 flow tube 4 by positioning the tip 15 of a pipette 16 above the continuous flow tube inlet 5 and dispensing about 10 pL of the liquid sample 2. The aqueous liquid sample 2 dispensed into the silicone oil 3 is substantially spherical in shape. Preferably the tip 15 of the pipette 16 remains in contact with the sphere of liquid sample 2 to assist in manoeuvring the sphere to the continuous flow tube inlet 5 and prevent it from "falling 10 away" to the walls of the reservoir 6. Since there is a flow of fluid carrier 3 into the continuous flow tube inlet 5 the sphere of liquid sample 2 can then be "touched off' into the inlet 5 (see Figure 3) from where it is drawn into the continuous flow tube 4 by the suction force applied to the outlet. As discussed above, whilst the fluid carrier 3 prevents contamination between 15 liquid samples 2 flowing through the continuous flow tube 4, the present Applicant has determined that the fluid carrier 3 should also be chosen to maintain the physical properties of the liquid sample 2. To explain, the fluid carrier 3 is preferably silicone oil having a viscosity of between about 5 to 50 centistokes and a density of about 0.98 g/cc thereby providing neutral buoyancy to the liquid sample 2. The Applicant has found that 20 the sphere of liquid sample 2 tends to "fall away" to the walls of the reservoir 6 and become adhered/lodged on the walls if the incorrect oil is used. Further, if the sphere descends too quickly it may be "caught" near the continuous flow tube inlet 5 and not be entirely drawn into the continuous flow tube 4. Referring now to Figure 4, a pair of sample tubes 17, 18 are provided in an 25 alternative port arrangement. The sample tubes 17, 18 are housed between a pair of spaced parallel plates 19 to define a rotatable stage 20. One of the sample tubes 17 is continuously replenished with fluid carrier 3 from a pump whilst the other sample tube 17 is free to receive a liquid sample 2 at atmospheric pressure. Once a liquid sample 2 is loaded into the sample tube 18 the tube 18 is then switched "in-line" by rotation of the 30 rotatable stage 20, thereby introducing the liquid sample 2 into the continuous flow tube 4. The sample tube 17 "replaced" by sample tube 18 (that has just been switched "in line") is thus available to receive a subsequent liquid sample 2. In an alternate configuration for high throughput applications, a plurality of continuous flow tubes 4 may be provided for simultaneously conducting a plurality of - 12 nucleic acid amplifications. The continuous flow tube inlets 5 are preferably spaced into an array within the reservoir 6. This embodiment is conducive for robotic sample handling whereby a robotic system can introduce a plurality of liquid samples 2 into the plurality of continuous flow tube inlets 5. Alternatively, the array of continuous flow 5 tubes may merge downstream in a parallel configuration into a single continuous flow tube 4 thereby defining a "parallel" manifold. In this embodiment the liquid samples 2 are loaded sequentially from one end of the manifold to the other thereby allowing the liquid samples 2 to be evenly spaced as they are drawn into and travel through the continuous flow tube 4. In other embodiments, the array of continuous flow tube 10 openings 5 may merge downstream into a single continuous flow tube 4 in series, thereby defining a "series" manifold. As will be appreciated, in this embodiment the liquid samples 2 may be loaded simultaneously. The sample port according to the present invention is easily adaptable to many types of continuous flow apparatus. Apart from the port itself, the only substantive 15 change to these existing apparatus is the provision of a suction force to the outlet of the continuous flow tube, if gravity feeding is insufficient, to draw the fluids therethrough rather than "pushing" the liquid stream through via high-pressure pumping. The suction force, where required, can be supplied by a standard vacuum pump or the like. The present invention will now be described with reference to the following 20 examples which should be considered in all respects as illustrative and non-restrictive. EXAMPLES Example 1: Atmospheric pressure sample port ("zero contamination" sample injector) Use of the sample port of the present invention enables an improved method for 25 contamination-free sample application as well as the use of standard pipette tips for injecting the sample that can be easily sterilised and, as required, each tip discarded after sample application. The sample port according to the present invention was fluidly engaged to ETFE (Tefzel) continuous flow tubing having internal diameter of 1.0 mm, external diameter 30 of 1.6 mm and length approx. 15 m. Silicon oil was trailed having a similar density to water and 5 centistokes viscosity. This oil was sufficient to flow through the tubing under the effects of gravity and the oil flowed at 100 pL/min when the injection port was approx. 50 cm higher than the outlet of the continuous flow tube. When silicon oil with P:\WPDOCS\RS\Corbcn\20417486 doc.-/06/209 - 13 a similar density to water and a 50 centistokes viscosity was trailed a vacuum of 20 kPa needed to be applied to the tube outlet to achieve a 200 pL/min flow rate. Commercial PCR buffers and TAQ's are supplied including a surfactant. This surfactant causes DNA to migrate from the aqueous phase to the oil phase during the flow 5 process. Running positive and negative PCR controls results in contamination in the negative sample in about 20 cycles after the positive (i.e. 1 in a million). This level of contamination is not acceptable for PCR applications as typically a I in a billion level of amplification is achieved. In a separate set of experiments each sample was loaded after a purge with 10 milliQ water as follows: 1) Load 5 tL water 2) Load 5 ltL sample 3) Repeat 1) By injecting pure water samples in between every PCR reaction sample (i.e. 15 "wash" fluid) contamination was not observed between a positive sample that amplified at cycle 3 and a negative sample that was run to 43 cycles. The samples injected were H 2 0 positive -H 2 0 - negative - 1-120 - positive ... etc). Therefore, water injection between each sample provides a contamination level of better than 1 in a billion. Although the invention has been described with reference to specific examples, 20 it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should riot be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or 25 information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. A sample port for introducing a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said port 5 comprising: a reservoir for continuously supplying said inlet with said fluid carrier, said reservoir adapted to maintain a substantially constant level of fluid carrier above said inlet and fluidly engageable with said inlet of said continuous flow tube such that, in use, said fluid carrier stream and said liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure and when said fluid 10 carrier is chosen such that its properties are sufficient to maintain the physical shape of the liquid sample introduced into said carrier.

2. A sample port according to claim 1, wherein the liquid sample is an aqueous sample.

3. A sample port according to claim 1 or claim 2, wherein the carrier fluid is a 15 hydrophobic liquid.

4. A sample port according to claim 3, wherein the hydrophobic liquid is an oil.

5. A sample port according to claim 4, wherein the oil is a silicon oil or a silicon based oil.

6. A method for introducing a volume of a liquid sample into a fluid carrier stream 20 flowing through a continuous flow tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said method comprising the steps of: providing a sample port according to any one of claims 1 to 5; fluidly engaging said inlet of said continuous flow tube with said reservoir; introducing said fluid carrier into said reservoir and introducing said liquid sample into said fluid carrier, 25 said fluid carrier chosen such that its properties are sufficient to maintain the physical shape of the liquid sample and such that said fluid carrier stream and said liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure.

7. A method for introducing a volume of a liquid sample into a fluid carrier stream 30 flowing through a continuous flow tube having an outlet and a common inlet into which said carrier stream and said liquid sample are both introduced, said method comprising the steps of: providing a port according to any one of claims 1 to 5; fluidly engaging said inlet of said continuous flow tube with said reservoir; introducing said fluid carrier into said reservoir; immersing a liquid sample dispenser into said fluid carrier contained in P:WPDOCS\GRS\Corbeu\20417486.doc-/6/2009 - 15 said reservoir; dispensing said liquid sample adjacent said inlet and optionally manoeuvring said dispensed liquid sample with said liquid sample dispenser such that said dispensed liquid sample is introduced into said inlet and drawn through said continuous flow tube, said fluid carrier chosen such that its properties are sufficient to maintain the physical shape of the liquid sample and such that said fluid carrier stream and said liquid sample are drawn through said continuous flow tube when said reservoir is at substantially atmospheric pressure.

8. A method according to claim 6 or 7 wherein the fluid carrier is drawn into the continuous flow tube by applying a suction force to the outlet.

9. A continuous flow apparatus comprising the sample port according to any one of claims 1 to 5.

10 A continuous flow apparatus according to claim 9, which is a thermocycling apparatus for performing nucleic acid amplification reactions.

11. A method of amplifying a nucleic acid in a PCR or LCR format using the continuous flow apparatus according to claim 10.

12. A sample port for introducing a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow tube substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.

13. A method for introducing a volume of a liquid sample into a fluid carrier stream flowing through a continuous flow tube substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.

14. A continuous flow apparatus substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.