Abstract:
In a preferred embodiment, a method of performing a reagent protocol using polymerase chain reaction, including: indexing patterns of reagent wells on a continuous basis through at least one step of reagent addition to the reagent wells; and then indexing the patterns of reagent wells on a continuous basis through a plurality of individual heat transfer stations, whereby at each of the individual heat transfer stations, the patterns of reagent wells are subjected to a unique temperature change to cause one amplification step, with the plurality of individual heat transfer stations providing total amplification required for the protocol.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation-in-part of co-pending application Ser. No. 09/425,070, filed Mar. 22, 1999, and titled TEMPERATURE CONTROL SYSTEM FOR POLYMERASE CHAIN REACTION, now U.S. Pat. No. 6,537,752, which is a continuation-in-part of application Ser. No. 09/198,018, filed Nov. 23, 1998, and titled ULTRA HIGH THROUGHPUT BIOASSAY SCREENING SYSTEM, which application claims the benefit of the filing dates of Provisional Patent Applications Nos. 60,067,895, filed Dec. 8, 1997, and titled ULTRA HIGH THROUGHPUT BIOASSAY SCREENING SYSTEM AND METHOD; 60/073,329, filed Feb. 2, 1998, and titled ULTRAHIGH THROUGHPUT BIOASSAY SYSTEM AND METHOD; and 60/095,497, filed Aug. 6, 1998, and titled USE OF CONTINUOUS CARRIER TAPE FOR POLYMERASE CHAIN REACTIONS, the disclosures of all which applications are incorporated by reference hereinto. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to polymerase chain reactions generally and, more particularly, but not by way of limitation, to a novel continuous polymerase chain reaction process having multiple temperature stations. 
     2. Background Art 
     In the field of genomics, and other disciplines using molecular biology, the polymerase chain reaction (PCR) protocol is essential. It is an amplification technique that utilizes three basic temperatures to amplify DNA. In such a protocol, the DNA is first brought to 96° Centigrade to denature the DNA, causing it to “unwind” from the standard double helix to single strands. The denaturing process requires exposure to 96° Centigrade for approximately 15 seconds. 
     Next in the protocol, the DNA is exposed to a temperature of 50-55° Centigrade to anneal the single strands, normally in the presence of defined primers. Again, approximately only 15 seconds at 50-55° Centigrade is required for annealing. The next temperature is 72° Centigrade. At this extension temperature, the two single strands form two double stranded helixes, thus resulting in a two-fold amplification. The extension temperature of 72° Centigrade is only required for 30 seconds. 
     The foregoing temperature cycling doubles the amount of DNA on each cycle. After 25 to 35 cycles, non-measurable quantities of DNA now become readily detectable because of the power of PCR and its exponential amplification. 
     The current state-of-the-art techniques for thermocycling comprise two basic methods. One is a batch method, whereby a group of PCR reaction plates is physically moved from one water temperature bath to another. The second, and more popular, method is the use of thermocycling instrumentation using Peltier thermoelectric devices to change the temperature of an individual PCR plate. 
     The Peltier thermoelectric device is clean and efficient; however, it process only one plate at a time. While the latter feature is an advantage for small operators, it is a disadvantage in high volume operations. High volume laboratories will have bench tops with many thermocyclers side by side. At a cost of $5,000-6,000 each, a considerable investment is required, particularly since the nature of genomic testing requires a high volume of testing. 
     Another disadvantage of the thermocycling instrument is the time required to move from one temperature to the next. At present, the popular Peltier devices can only change temperature at a rate of about 3 Centigrade degrees per second. The change from 96° Centigrade to 50° Centigrade requires 15 seconds transient time plus the 15 seconds at the annealing temperature. From 50° Centigrade to 72° Centigrade requires 7 seconds transient time plus the 30 second extension time. From 72° Centigrade to 96° Centigrade requires 8 seconds. Thus, for the 60 seconds of protocol time, an additional 30 seconds is required for transient time. This adds 50 percent to the overall time cycle. While insignificant on a single cycle, the time is an additional 12 minutes per plate on a 25 cycle protocol and 17 minutes per plate on a 35 cycle protocol. 
     The batch method of inserting a stack of plates into separate water baths decreases the temperature transient time. While the batch method is suitable for batches of large numbers of plates, the set up and handling time makes running small batches less attractive. 
     Accordingly, it is a principal object of the present invention to provide a PCR process that greatly reduces temperature transient times. 
     It is a further object of the invention to provide such a process that is economical for either a small or a large number of DNA samples. 
     It is another object of the invention to provide such a process that is easily implemented. 
     A further object of the invention is to provide such a process that is continuous. 
     Other objects of the present invention, as well as particular features, elements, and advantages thereof, will be elucidated in, or be apparent from, the following description and the accompanying drawing figures. 
     SUMMARY OF THE INVENTION 
     The present invention achieves the above objects, among others, by providing, in a preferred embodiment, a method of performing a reagent protocol using polymerase chain reaction, comprising: indexing patterns of reagent wells on a continuous basis through at least one step of reagent addition to said reagent wells; and then indexing said patterns of reagent wells on a continuous basis through a plurality of individual heat transfer stations, whereby at each of said individual heat transfer stations, said patterns of reagent wells are subjected to a unique temperature change to cause one amplification step, with said plurality of individual heat transfer stations providing total amplification required for said protocol. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     Understanding of the present invention and the various aspects thereof will be facilitated by reference to the accompanying drawing figures, submitted for purposes of illustration only and not intended to define the scope of the invention, on which: 
     FIG. 1 is a fragmentary, top plan view of a carrier tape used in the present invention. 
     FIG. 2 is a fragmentary, schematic side elevational view of a continuous PCR processing line according to the present invention. 
     FIG. 3 is a fragmentary, top plan view of pipettor heads servicing the processing line of FIG.  1 . 
     FIG. 4 is a fragmentary, side elevational view of a heat exchanger used in the present invention. 
     FIG. 5 a flow diagram of the temperature control system of the present invention. 
     FIG. 6 is a fragmentary, side elevational view of the mechanism for removal of a heat seal material. 
     FIG. 7 is a top plan view, partially cut-away, illustrating an alternative embodiment of sealing carrier tape used in the present invention. 
     FIG. 8 is an enlarged, fragmentary, side elevational view, of a heat seal useful in the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Reference should now be made to the drawing figures, on which similar or identical elements are given consistent identifying numerals throughout the various figures thereof, and on which parenthetical references to figure numbers direct the reader to the view(s) on which the element(s) being described is (are) best seen, although the element(s) may be seen also on other views. 
     FIG. 1 illustrates a sprocket-driven, multi-well carrier tape, generally indicated by the reference numeral  20 . The above-referenced applications describe, in further detail, carrier tape  20  and uses thereof. 
     Carrier tape  20  includes a substrate web  30  in which is formed a plurality of 10-microliter reagent wells, as at  32 , embossed into the web, or thermoformed in the web, in patterns of 16×24 matrixes, with the wells on 4.5 mm centers. Such a pattern is indicated by the reference numeral  40 . A plurality of vent holes, as at  50 , may be provided through substrate web  30  to assist in effecting a seal between the substrate web and a seal layer (not shown on FIG. 1) when a seal layer is used. A series of holes comprising a binary code of identification, such as holes  60 , may be formed through substrate web  30  in order to identify pattern  40 . Alternatively, or in addition, a bar code  62  and/or humanly readable indicia  64  may be provided for pattern identification. Other suitable identifying indicia may be provided as well. Precision sprocket holes, as at  70 , are provided spaced along either edge of carrier tape  20  to provide a means of transporting patterns  40  from one location to the next within a processing line. This enables a positive, position-controlled, indexing drive system. The drive system may, for example, be walking beams, geneva motions, electronic stepper drives, or pneumatic indexing mechanisms. 
     Carrier tape  20  may be constructed of one of several thermoplastic materials. Polypropylene is a satisfactory choice where there is no requirement that the material be clear. Where clearness is a requirement, polycarbonate provides a suitable clear material that facilitates optical readout of the final test results. Pattern  40  of 384 wells is a common format used in biotechnology. 
     Carrier tape  20  starts with a thickness of 20 mils. When wells  32  are formed, the wall thickness of each well decreases to a thickness of approximately 2 mils. This thin wall around the contents of a well provides a minimal thermal barrier to heat transfer. 
     FIG. 2 illustrates a PCR processing line, generally indicated by the reference numeral  100 . Processing line  100  includes a payoff reel  110  that supplies virgin carrier tape  20  (FIG. 1) to the processing line. An intermittent motion indexing drive  120  engages sprocket drive holes  70  (FIG. 1) and advances carrier tape  20  precisely one carrier tape pattern  40 . The indexing motion can be derived one of several ways. If can be stepper motor, cam drive, walking beam, geneva motions, or reciprocating air cylinders. In addition to indexing carrier tape  20  forward, indexing mechanism  120  clamps the carrier tape to the indexing mechanism so as to provide a positive position controlled drive system. 
     A punch mechanism  124  punches 8-bit binary code  60  (FIG. 1) between patterns. Binary code  60  provides a positive sample identification system. This code can be read at subsequent stations, or processing equipment, by contact fingers, air pressure jets, or photometric means. Punch mechanism  124  may alternatively, or in addition, include a bar code printer. 
     With reference primarily to FIG. 3, carrier tape  20  is next indexed such that pattern  40  (FIG. 1) is moved under a first transfer station, generally indicated by the reference numeral  130 . First transfer station  130  includes an identifying indicia reader  132  and a 384-well pipettor head  140  mounted on a Y-axis traverse  142  to allow the pipettor head to aspirate or dispense at either of the positions shown in solid or broken lines. Y-axis traverse  142  also permits pipettor head  140  to move to tip washing station  150 . A separate Z-axis traverse (not shown) built into pipettor head  140  allows vertical motion to reach three stations. An infeed plate stacker  160  can feed sample trays in the 384-well format (not shown) down onto an X-axis traverse  162  which can move the plate for access by pipettor head  140 . Following that, X-axis traverse  162  can then transport the plate to an output plate stacker  170 . 
     A typical operating sequence would be to move a 384-well microplate from infeed stacker  160  down to X-axis traverse that would transport the microplate to pipettor head  140 . Pipettor head  140  (solid position) would then aspirate 384 aliquots from the microplate and dispense (broken lines) the 384 samples onto pattern  40 . Pipettor head  140  would then traverse to tip washing station  150  to wash the tips (not shown) of the pipettor head. Concurrently with the later motion, X-axis traverse  162  would move the used microplate to outfeed stacker  170  and retrieve the next microplate from infeed stacker  160 . The cycle sequence would then repeat for the next indexed pattern on carrier tape  20 . 
     Continuing to refer primarily to FIG. 3, following first transfer station  130  is a second transfer station generally indicated by the reference numeral  180 . Second transfer station  180  is identical to first transfer station  130  and includes an identifying indicia reader  182  and a 384-well pipettor head  190 , Y-axis and X-axis traverses  192  and  194 , respectively, an infeed stacker  196 , and an outfeed stacker  198 . 
     Referring now again primarily to FIG. 2, in a typical operation, DNA samples would be added to carrier tape  20  at first transfer station  130  and primer/master mix would be added to the carrier tape at second transfer station  180 . 
     Binary code and/or bar code readers  132  and  182  at, respectively, first and second transfer stations  130  and  180  read the identification of incoming microplate samples. These numbers are tied to the carrier tape pattern number in a database to maintain a sample audit trail. 
     Continuing to refer to FIG. 2, following the addition of all reagents, carrier tape  20  is sealed. A payoff reel  210  contains a heat seal top covering  212  which is paid out over an idler roller  214  and heat sealed to carrier tape  20  at sealing station  216 . At sealing station  216 , a heat seal head closes, sealing top covering  212  to carrier tape  20 . 
     As indicated on FIG. 2, carrier tape  20  is now indexed through the PCR section of processing line  100 , the PCR section being indicated generally by the reference numeral  230 . PCR section  230  consists of a plurality of identical thermal transfer stations, as at  240 , with each station providing one set of temperature changes to the reagents in the pattern, resulting in one amplification cycle for the reagents. The number of thermal transfer stations  240  provided is dependent on how many PCR cycles are required and, ordinarily, there are at least as many individual thermal transfer stations as there are amplification cycles required by the protocol. This requires enough thermal transfer stations in processing line  100  to handle the maximum number of amplification cycles for which the processing line is designed. A typical processing line  100  may have 35 or more thermal transfer stations  240 , although all thermal transfer stations may not be used for all protocols. If 35 thermal transfer stations  240  are provided and a particular protocol requires only 25 PCR cycles, then the remaining 10 thermal transfer stations would not be activated. The unused thermal transfer stations  240  do not temperature cycle and in essence are bypassed, although carrier tape  20  will index through them. 
     Referring to FIG. 4, thermal transfer station  240  includes a small liquid chamber  260  that is clamped to the bottom side of carrier tape  20  around pattern  40  (FIG.  1 ). Chamber  260  is created between a backup plate  270  and a heat exchange reservoir  272 . This clamping motion may be provided by one of several means for each thermal transfer station  240 , such as an air cylinder  274 , or it may be a common mechanical motion, clamping all thermal transfer stations in common. An elastomeric gasket  280  effects a liquid tight seal between the upper edges of heat exchange reservoir  272  and the bottom of carrier tape  20 . The heat transfer medium in heat exchange reservoir  272  is in direct contact with reagent wells  32  protruding from the bottom of carrier tape  20 . Thus, the heat transfer is by intimate conduction. This, combined with the thin walls of reagent wells  32 , provides a very fast heat transfer to the PCR components within the wells. 
     Backup plate  270  and heat exchange reservoir  272  are fabricated from a heat insulating material such as polypropylene to minimize the heat loss through conduction by these elements on the quick changing liquid temperature within each heat transfer station  240 . 
     FIG. 5 illustrates the temperature control system for thermal transfer stations  240  and includes, in the system shown, three reservoirs  280 ,  282 , and  284  which may contain water, for example, as the heat transfer medium. For the protocol described above, reservoir  280  would contain water at the denaturing temperature of 95° Centigrade (“T 1 ”), reservoir  282  would contain water at the annealing temperature of 55° Centigrade (“T 2 ”), and reservoir  284  would contain water at the extension temperature of 72° Centigrade (“T 3 ”). Each of reservoirs  280 ,  282 , and  284  has a heating element  290 , a cooling coil  292 , and a proportional/integral/derivative controller  294 , the latter being able to cycle between heating and cooling to hold precise temperatures within the reservoirs. 
     Water from reservoirs  280 ,  282 , and  284  is fed to thermal transfer stations  240  by means of, respectively, circulating pumps  300 ,  302 , and  304  through feed lines  310 ,  312 , and  314  and is returned to the reservoirs, respectively, through return lines  320 ,  322 , and  324 . 
     Each heat transfer station  240  has its own series of feed and return solenoid valves, as at, respectively,  330  and  332 . When the program requires, the extension temperature T 1 , valves  330  and  332  open and water at temperature T 1  flows through heat transfer station  240 , effecting heat transfer to the reagents contained in wells  32  (FIG.  4 ). When a fast response temperature sensor  340  located in the return from heat transfer station  240  reaches a predetermined temperature, it initiates the timing sequence for that temperature at that heat transfer station. When the time for T 1  expires, the feed and return valves for T 1  close and the feed and return valves for T 2  open. This sequence is repeated for T 3 . When the T 3  cycle time expires, the feed valve closes, but the return valve stays open, allowing some drainage from the heat transfer station. 
     When all heat transfer stations  240  have completed their temperature cycles, the heat transfer stations are opened enough to allow carrier tape  20  to index one station. The entire sequence then repeats for the next index. Each index equates with a change of temperature through T 1 , T 2 , and T 3  for each station. These amplification cycles occur simultaneously at all heat transfer stations  240  being used in the protocol. After the first pattern  40  has progressed through all heat transfer stations  240 , a completed pattern of reagents is presented on each indexing of the system. Thus, if the index rate is one index every 1.5 minutes, a complete set of samples will be completed thereafter every 1.5 minutes. 
     Reference should be made again to FIG.  2 . Following the amplification by PCR, there are several options for post processing. As shown on FIG. 2, the processed carrier tape  20  may be indexed directly into a fluorescent reader  400 . In this case, the reading is made without removing seal layer  212 . A transparent seal material is used for seal layer  212  and the reading of the well contents is made through the seal layer. As carrier tape  20  exits reader  400 , it may be wound on a take up reel  420  driven by a torque motor  422 . Another option is to cut carrier tape  20  into pieces for disposal in a container (not shown). 
     While a heat seal material provides a more secure seal, the use thereof does require additional complexity for its removal, as is shown on FIG.  6 . Seal layer  212  is a peelable seal material bonded to carrier tape  20  by means similar to that used for lids in the food industry. At the time of forward index motion of carrier tape  20 , a heated roll  450  is brought into contact with the top of seal layer  212 . This provides a line of high temperature across seal layer  212  which, at the point of contact, softens the seal layer. Tension is applied to seal layer  212  by take up reel  460  driven by torque motor  462 . This separates seal layer  212  from carrier tape  20  and draws the seal layer over guide roller  470  and winds the seal layer upon take up reel  460 . When the forward index motion of carrier tape  20  stops, heated roll  450  swings away from contact, so as not to burn through when the carrier tape is stopped. Removal of seal layer  212  permits access to the contents of reagent wells  32  (FIG.  1 ). The contents of wells  32  can then be aspirated by another pipettor station and transferred to another element for further use or processing. 
     Some applications may not require sealing the top of carrier tape  20  and the clamping action at individual heat transfer stations  240  (FIG. 2) may be sufficient to prevent evaporation and cross contamination. 
     Referring to FIG. 7, another alternative is to use a seal layer  212 ′ on a carrier tape  20 ′, the seal layer having a printed pressure sensitive adhesive pattern  500  that would only bond around the perimeter of well patterns  240 ′. The area of seal layer  212 ′ over well patterns  240 ′ would not have adhesive. This arrangement facilitates removal of seal layer  212 ′ and eliminates any cross contamination between well patterns  240 ′. Clamping pressure at each heat transfer station  240  prevents cross contamination between wells  32  (FIG. 1) within well patterns  240 ′. 
     FIG. 8 illustrates a heat seal layer, generally indicated by the reference numeral  600 . Heat seal layer  600  provides a more secure sealing method and consists of a two part construction comprising a top layer  610  with high melting point and strength bonded to a low temperature sealing layer  620 . A typical heat seal layer  600  would be a bifilm combination of polyester/ethylene vinyl acetate or of an aluminum foil with a heat seal coating. The heat seal material or coating would bond to the carrier tape, creating a liquid tight seal around the perimeter of each individual reagent well. 
     In the embodiments of the present invention described above, it will be recognized that individual elements and/or features thereof are not necessarily limited to a particular embodiment but, where applicable, are interchangeable and can be used in any selected embodiment even though such may not be specifically shown. 
     Terms such as “upper”, “lower”, “inner”, “outer”, “inwardly”, “outwardly”, and the like, when used herein, refer to the positions of the respective elements shown on the accompanying drawing figures and the present invention is not necessarily limited to such positions. 
     It will thus be seen that the objects set forth above, among those elucidated in, or made apparent from, the preceding description, are efficiently attained and, since certain changes may be made in the above construction and/or method without departing from the scope of the invention, it is intended that all matter contained in the above description or shown on the accompanying drawing figures shall be interpreted as illustrative only and not in a limiting sense. 
     It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.