Patent Application: US-60281500-A

Abstract:
the polymerase chain reaction is one of the most widely used techniques in molecular biology . in general , most thermocyclers which automate the pcr nucleic acid amplification process rely upon programmable heat blocks with a large thermal mass . consequently , most of the time in an automated pcr cycle is spent non - productively in transition between denaturation , annealing , and elongation temperatures . recently , much faster hot - air thermocyclers have been constructed which shorten these transition times , allowing 30 cycles of pcr in 10 to 30 minutes . while elegant in principle , the design of these systems is not optimal . air is a relatively poor heat transfer medium ; and the operation of a single heat / reaction chamber at atmospheric pressure is inherently slow . much faster thermocyclers can be constructed using pressurized gas delivered to a thermostated reaction chamber by computer - controlled electronic valves . a novel process , high - speed gas phase pcr , is described . this process has been successfully automated using a novel thermocycing device , which has been successfully to amplify dna from picogram to microgram amounts in ˜ 1 to 5 minutes .

Description:
the present invention will be described as it applies to an exemplary embodiment . it is not intended that the present invention be limited to the described embodiment . it is intended that the invention cover all modifications and alternatives which may be included within the spirit and scope of the invention . diligent development of this invention has proceeded in three stages : ( 1 ) design of the pressurized gas thermocycler , ( 2 ) fabrication and optimization of the device , and ( 3 ) use of the device and process in high - speed pcr amplification experiments . we wished to build a pressurized gas thermocycler which would carry out high - speed pcr amplification of dna by injecting hot or cold gas into a thermostated reaction chamber under the control of fast (& lt ; 15 msec / cycle ) electronic valves , digital relays , and a microprocessor controller . ideally , this device would contain a minimum number of moving parts and be compatible with optics used for detection of fluorescent dye - labeled dna ( higuchi et al ., 1992 ; haugland , 1996 ). in particular , we designed a device in which the temperature could be rapidly changed by injecting hot and cold pressurized gas into a reaction chamber under electronic control , using digitally programmable “ hot gas ” and “ cold gas ” valves . the pressure in the reaction chamber is maintained at a relatively constant value by means of a mechanical relief valve , although the temperature can be changed upon demand . reaction samples ( 10 to 20 microliters in volume ) are sealed in thin - walled capillary tubes as described by wittwer and garling ( 1991 ). therefore , although the pressure inside the reaction chamber varies , the pressure in the capillary tubes remains unchanged . a pressurized gas thermocycler differs from a hot - air thermocycler ( u . s . pat . no . 5 , 455 , 175 to wittwer et al .) in several important respects , as shown in fig2 . in the hot - air thermocycler ( fig2 a ), a single reaction chamber with a heat lamp is used to heat air and to carry out pcr reaction ( heat chamber = rxn chamber ). in the pressurized gas thermocycler ( fig2 b ), three separate chambers are used : a heating chamber , a cold gas supply chamber , and a reaction chamber . in order to heat samples in the reaction chamber , pre - heated gas from the heat chamber is delivered under pressure to the reaction chamber by electronic valve v h ( hot gas valve 20 ). in order to cool samples in the reaction chamber , cold gas is delivered under pressure to the reaction chamber by electronic valve v c ( cold gas valve 21 ). a mechanical relief valve v r ( 18 in fig3 a ) allows exit of pressurized gas from the reaction chamber . reference will now be made to the drawings wherein like structures will be provided with like reference designations . schematic drawings of the pressurized gas thermocycler and its reaction chamber are shown in fig3 a , 3 b , and 3 c . as seen in fig3 a , the thermal cycling device 10 includes a reaction chamber made of insulating ( low k ) material , generally designated at 23 , which is adapted to accept samples to be introduced through a closing cap 24 . preferably , reaction chamber 23 has k value of less than 0 . 5 watts / centimeter - degree k . reaction chamber 23 can be comprised of a number of different materials , such as stainless steel or titanium . polyurethane or polymethane plastics with ceramic binders , sold under trade names such as vespel , torlon , and butterboard ( which is manufactured by golden west of calif . ), can also be used as a reaction chamber 23 material . cold gas supply 12 is separated from reaction chamber 23 by a one - way check valve 13 ( available from linweld of lincoln , nebr .) and an electronic valve 21 ( a 5 vdc valve manufactured by spartan scientific of ohio ) which is actuated by a digital relay operated by controller 29 . controller 29 can be a microprocess or programmable logic controller ( plc ), such as a micro - 485 manufactured by blue earth of mankato , minn . hot gas supply 11 is connected to a process heater 14 ( 500 watt process heater manufactured by hotwatt of danvers , mass .) using a one - way check valve 13 . process heater 14 is actuated by a heavy - duty relay operated by controller 29 according to thermal sensor 15 ( manufactured by physitemp of new jersey ) which monitors the temperature of the heated gas . hot gas exiting the heater 14 flows from connecting cross 16 through an electronic valve 20 ( operated by controller 29 ) into the reaction chamber 23 . the process heater requires a continuous flow of gas whenever valves 20 and 22 are closed ; therefore a mechanical relief valve 17 ( 30 p . s . i .) is connected to the cross 16 . the system pressure and flow of hot gas into the reaction chamber 23 can be reduced by opening the throttle valve 22 ( operated by controller 29 ), which is connected to a mechanical relief valve 19 . mechanical relief valves 18 and 19 have the same cracking pressure ( 20 p . s . i .). therefore , when valve 22 is open , the hot gas flow rate and pressure entering the chamber 23 are reduced . as shown in fig3 b , valves 20 and 21 can be opened or closed upon demand , using controller 29 , based upon the temperature in the reaction chamber 23 measured by the thermocouple 30 . as shown in fig3 c , the reaction chamber 23 contains a central cavity 27 , which contains reaction samples sealed in thin - walled capillary tubes . hot gas enters the chamber from hot gas valve port 25 , passes through flow conditioner 28 into the central cavity 27 . cold gas enters the chamber from cold gas valve port 26 , passes through flow conditioner 28 into central cavity 27 . the flow conditioner 28 helps maintain the desired temperature of the reaction chamber 23 . gas exits the reaction chamber 23 through mechanical relief valve 18 . it is preferable to be able to hold temperature in the reaction chamber to within about ± 1 ° c . for accurate primer : dna annealing and taq polymerase elongation . in order to do so , a third valve was added , a so - called throttle valve ( 22 in fig3 a ), which made it possible to drop the system pressure during elongation steps . this tri - valve device ( hot gas valve , cold gas valve , throttle valve ) was found to give excellent thermal control . the tri - valve pressurized gas thermocycler was tested with various combinations of hot and cold supply gases . in general , pressurized air is available , inexpensive , and easy to control . on the other hand , pressurized helium gas is a superior heat transfer gas ( fig1 ), but is more problematic with respect to thermal control ( fig8 b ). operation of the pressurized gas therrnocycler is outlined schematically in fig9 . there are basically three operating modes , which are controlled by activating relays wired to the valves : ( 1 ) heating the reaction chamber , ( 2 ) cooling the reaction chamber , and ( 3 ) holding the temperature of the reaction chamber . in order to increase the temperature of the reaction chamber 23 , the hot gas valve 20 is opened , so that hot pressurized gas is delivered to the chamber . the cold gas valve 21 and throttle valve 22 remain closed . in order to reduce the temperature of the chamber , the hot gas valve 20 is closed , the cold gas valve 21 is open , and the throttle valve 22 is closed . during the elongation step of the pcr process , when the temperature is held near 72 ° c ., the cold gas valve 21 is closed , the throttle valve 22 regulating the system pressure and gas flow rate is open and the hot gas valve 20 is open or closed upon demand based on the temperature measured by the thermal sensor 30 located in the reaction chamber 23 . this valve configuration provides superior thermal control for holding the temperature near a preset value . the opening and closing of hot , cold , and throttle valves is controlled by electrical signals from the system controller to electrical relays which actuate the valves . the system control software determines the opening and closing of these valves . when different “ hot ” or “ cold ” supply gases are employed , modified software is utilized , since gas flow and mixing in the chamber varies , depending upon the gas mixtures chosen . it is convenient to use either pressurized air or helium as a “ hot ” gas ; and to use air or co 2 as a “ cold ” gas ; but a variety of different gases can be employed . it is neither necessary nor optimal to use the same gas to heat , cool , or hold the temperature of the chamber . as shown below ( fig5 a , 6 , 7 , and 8 a ) high - speed gas phase pcr amplification of dna can be carried out using pressurized air ( or helium ) as a ‘ hot ’ gas ; and pressurized co 2 as a ‘ cold ’ gas . thirty cycles of pcr amplification can be achieved in 78 to 335 seconds , depending upon the heat transfer gases chosen . in the examples described below , amplified dna was detected using a relatively slow (˜ 1 hour ) process : gel electrophoresis followed by ethidium bromide staining , a standard method of dna detection known to those practiced in the art . however , gas phase pcr is compatible with rapid on - line fluorescent dye - based detection ( higuchi et al ., 1992 ) using dyes such as sybr green , which bind selectively to double - stranded dna ( haugland , 1996 ). in principle , the double - stranded dna reaction products of high - speed gas phase pcr can be rapidly detected on - line using reporter dyes like sybr green and inexpensive photodiode detectors . for example , a pressurized gas thermnocycler could be fitted with photodiode detectors and either a light - emitting diode ( led ) or laser diode dye excitation source . such optics would allow amplification / detection of dna on an unprecedented time scale . initial gas phase pcr experiments were carried out with compressed air as a “ hot ” gas and co 2 as a “ cold ” gas . this configuration was not the fastest possible , but was convenient and did not require active refrigeration of the “ cold ” gas . the first successful dna amplification experiments were carried out using pressurized air ( 36 p . s . i .) as a “ hot ” gas and pressurized co 2 (˜ 40 p . s . i .) as a “ cold ” gas in the tri - valve machine ( fig3 a ). four model dna templates of different lengths were pcr - armplified in separate 10 μl reactions , which were carried out in thin - walled glass capillary tubes : ( a ) a 91 b . p . e . coli o157 : h7 stx amplicon , ( b ) a 333 b . p . λ ‘ d ’ gene amplicon , ( c ) a 364 b . p human platelet antigen hpa - 4 amplicon , and ( d ) a human β - globin 536 b . p . amplicon were amplified using 30 cycles of [ 0 sec 92 ° c ./ 0 sec 55 ° c ./ 5 sec 72 ° c .] with pressurized air as a hot gas and co 2 cooling . gas phase pcr reactions ( 10 μl ) were carried out thin - walled glass capillary tubes containing 50 mm tris ( ph 8 . 5 at 25 ° c . ), 250 μg / ml bsa , 3 mm mgcl 2 , 0 . 2 mm dntps , 50 pmol of forward and reverse primers , 20 picograms of template dna , and 5 u of taq polymerase ( promega , madison , wis .). after amplification , reaction products were separated on 3 % metaphor agarose gels w / etbr staining . molecular wt . markers are 67 - 501 b . p . long ( puc19 / mspi dna fragments ). as shown in fig5 a , all four amplicons were amplified in high yield using gas phase pcr . the expected 536 b . p . β - globin gene , 364 b . p . hpa - 4 allele , 333 b . p . phage λ ‘ d ’ gene , and 91 b . p . e . coli o157 : h7 stx amplicons were amplified through 30 cycles of [ 0 sec 92 ° c . ( denaturation )/ 0 sec 55 ° c . ( annealing )/ 5 sec 72 ° c . ( elongation )]. a low background of non - specific amplification products was observed in all four amplification reactions . in the case of the λ ‘ d ’ gene amplicon , an unusually high yield was also observed ( lane b ). a temperature vs . time profile of this 335 second gas phase pcr experiment showed considerable improvement in performance over any previously reported thermocycler or thermocycling method ( fig4 and 5 b ). software refinements , 30 gas phase pcr cycles in 2 : 48 ( 168 seconds ) at this point , it was clear that the tri - valve apparatus was a functional therrnocycler . not only was it very fast , but it exhibited good thermal control (± 1 ° c .). however , its performance was limited by its software , which was written in basic code . every time it needed to execute commands in response to thermal changes in the reaction chamber or heat pipe , it needed to translate from basic ( interpreted program ) to assembly code ( compiled program ). a substantial amount of time (˜ 1 sec / cycle ) was lost due to this software limitation . in particular , during 30 cycles of gas phase pcr amplification , over 30 seconds of time were nonproductively lost . consequently , the system control software was re - written in assembly code for faster operation . this modification resulted in much faster gas phase pcr , as shown in fig6 . thirty cycles of [ 0 sec 92 ° c ./ 1 sec 55 ° c ./ 1 sec 72 ° c .] were carried out using 36 of pressurized air with co 2 cooling . gas phase pcr reactions ( 10 μl ) were carried out in thin - walled glass capillary tubes containing 50 mm tris ( ph 8 . 5 at 25 ° c . ), 250 μg / ml bsa , 3 mm mgcl 2 , 0 . 2 mm dntps , 50 pmol of forward and reverse primers , 20 picograms of template e . coli o157 : h7 dna , and 5 u of polymerase . after amplification , reaction products were separated on 3 % metaphor agarose gels with etbr staining . molecular wt . markers are 67 - 501 b . p . puc19 / mspi dna fragments . lane ( a ) 5 units taq pol , ( b ) 5 units taqz pol ( takara shuzo ). fig6 shows that 30 cycles of gas phase pcr can be carried out in less than 3 minutes ; and that a considerable improvement in performance was achieved by writing instrument commands in assembly code . fig6 also demonstrates that an infectious disease agent ( e . coli o157 : h7 ) could be detected using gas conditions suitable for routine analysis in many laboratories ( pressurized air , bottled co 2 cooling ). either taq pol ( promega ) or a modified taq polymerase ( takara z - taq ™ pol , takara shuzo ltd ) was suitable for rapid (& lt ; 3 minute ) gas phase pcr . even faster gas phase pcr amplification was achieved using short (& lt ; 100 b . p .) amplicons . at a taq polymerase elongation rate of ≧ 80 nucleotides / sec , it was expected that ˜ 1 sec / cycle spent during the transitions a - e + e - d ( fig4 a ) would be more than sufficient to copy a short 85 b . p . dna template using a 30 mer pcr primer ( t m ˜ 65 ° c .). accordingly , experiments were carried out with pressurized air heating / co 2 cooling , using a relatively short ( 85 b . p .) amplicon from the e . coli o157 : h7 stx verotoxin gene . gas phase pcr reactions ( 10 μl ) were carried out with 36 p . s . i . of pressurized air and co 2 cooling in thin - walled glass capillary tubes containing 50 mm tris ( ph 8 . 5 at 25 ° c . ), 250 μg / ml bsa , 3 mm mgcl 2 , 0 . 2 mm dntps , 50 pmol of forward and reverse primers , 20 picograms of template e . coli o157 : h7 dna , and 5 u of taq polymerase ( promega ). after 30 cycles of amplification , dnas were separated on 3 % metaphor agarose followed by etbr staining . m . w . markers are 67 - 501 b . p . long puc19 / mspi digest . fig7 shows that , even with suboptimal pressurized air heating and co 2 cooling , a short 85 b . p . e . coli o157 : h7 stx amplicon could be amplified in high yield in & lt ; 2 minutes . based upon the high heat transfer coefficient of helium gas ( ubbink , 1947 ; bosworth , 1952 ; fig1 ), it was hypothesized that even faster thermocycling would be possible if helium was used rather than air as a “ hot ” gas . a small ( 85 b . p .) amplicon from the e . coli o157 : h7 stx gene was pcr - amplified using “ hot ” pressurized helium gas and co 2 cooling . in order to further reduce the thermocycling time , primer lengths were increased to 30 mers , so that higher annealing temperatures ( 62 ° c . to 63 ° c .) could be employed . in addition , the dna denaturation temperatures were slightly reduced ( 86 ° c . to 89 ° c .) from those used in previous experiments . three different high - speed gas phase thermocycling protocols were employed : ( a ) [ 0 sec 89 ° c ./ 0 sec 62 ° c ./ 0 sec 72 ° c . ]; ( b ) [ 0 sec 87 ° c ./ 0 sec 62 ° c ./ 0 sec 72 ° c . ]; and ( c ) [ 0 sec 86 ° c ./ 0 sec 63 ° c ./ 0 sec 72 ° c .]. pcr reactions ( 10 μl ) were carried out in thin - walled glass capillary tubes containing 50 mm tris ( ph 8 . 5 at 25 ° c . ), 250 μg / ml bsa , 3 mm mgcl 2 , 0 . 2 mm dntps , 50 pmol of forward and reverse primers , 20 picograms of e . coli o157 : h7 dna , and 5 u of taq polymerase ( promega , madison , wis .). after 30 cycles of [ 0 sec 89 ° c ./ 0 sec 62 ° c ./ 0 sec 72 ° c . ]; ( b ) [ 0 sec 87 ° c . 0 sec 62 ° c ./ 0 sec 72 ° c . ]; and ( c ) [ 0 sec 86 ° c ./ 0 sec 63 ° c ./ 0 sec 72 ° c . ], reaction products were separated on 3 % agarose gels with etbr staining . molecular weight markers are 67 - 501 b . p . long ( puc19 / mspi digest ). as shown in fig8 a , a high yield of the expected 85 b . p . stx amplicon was seen in all three helium gas phase pcr reactions . fig8 a and 8 b demonstrate the three fastest dna amplification reactions which have ever been carried out . the expected 85 b . p . e . coli o157 : h7 stx amplicon was amplified in all three reactions : ( a ) 1 : 25 = 85 seconds , ( b ) 1 : 21 = 81 seconds , ( c ) 1 : 18 = 78 seconds . this experiment also shows that . thermocycling was considerably faster when pressurized helium gas was used rather than air . high - speed gas phase pcr also resulted in a very low background of non - specific “ haze ” or false reaction products . presumably , false priming or elongation are rare when such fast thermocycling parameters are used ; there is simply no time for spurious reaction products to accumulate . the above results can be summarized as follows : ( i ) a novel process , high - speed gas phase pcr , has been developed , which allows rapid amplification of dna . ( ii ) a pressurized gas thermocycling apparatus has been constructed , which allows automation of the pressurized gas pcr process . ( iii ) amplicons ranging in size from 85 b . p . to 536 b . p . have been successfully amplified . ( iv ) dna from a heritable human disease gene ( human platelet antigen hpa - 4 allele ) and an infectious disease agent ( e . coli o157 : h7 ) were amplified in high yield . ( v ) the background of non - specific pcr reaction products was extremely low . ( vi ) thermocycling was faster when hot , pressurized helium gas was used rather than hot , pressurized air . ( vii ) in our fastest dna amplification experiments , a short 85 b . p . amplicon was amplified through 30 cycles in 78 seconds . to put the speed of pressurized gas thermocyclers in perspective , the fastest experimental thermocyclers built by lawrence livermore national laboratory ( northrup et al ., 1998 ), the university of pittsburgh ( oda et al ., 1998 ) , and the university of washington ( friedman and meldrum , 1998 ) require 8 . 5 to 20 minutes for 30 cycles of dna amplification . for example , oda et al . ( 1998 ) described “ ultrafast pcr ” in which “ cycle times as fast as 17 seconds could be achieved .” the pressurized gas device shown in fig3 a requires less than 2 . 7 seconds / cycle ; and the reaction yield is higher ( see fig5 a , 6 , 7 and 8 a ). in addition , the infrared heating method used by oda et al . is incompatible with fluorescent optical detection using & gt ; 450 nm dyes and photodiode detectors . kopp et al . ( 1998 ) have described a miniature continuous - flow pcr device was able to amplify a 176 b . p . dna fragment through 20 cycles in 3 to 4 minutes . however , ˜ 10 8 copies of template dna were required as starting material ( zorbas , 1999 ). extrapolating from the data of kopp et al . ( 1998 ), 30 cycles of pcr amplification would require 4 . 5 to 6 minutes ; and the yield of amplified dna is orders of magnitude lower than that obtained using the pressurized gas device shown in fig3 a . the fastest commercially available thermocycler is manufactured by the boehringer - mannheim division of roche in germany , under license from idaho technology ( u . s . pat . no . 5 , 455 , 175 to wittwer et al .). this hot - air thermocycler requires about 9 . 5 minutes for 30 cycles of amplification of the 536 base pair β - globin dna fragment . however ,. with its on - line detection optics attached , the lightcycler ™ requires about 30 minutes for 30 cycles of pcr . as shown in fig8 b , a pressurized gas pcr process can be used to amplify dna ˜ 10 6 - fold in as little as 78 seconds . the high - speed pressurized gas thermocycling process is & gt ; 20 times faster than the roche machine and is compatible with on - line fluorescent dye - based dna detection optics . a general description of the present invention as well as a preferred embodiment has been set forth above . those skilled in the art will recognize and be able to practice additional variations in the methods and devices described which fall within the teachings of this invention . accordingly , all such modifications and additions are deemed to be within the scope of the invention which is to be limited only by the claims appended hereto . azbel d ( 1984 ) fundamentals of heat transfer for process engineering , noves publications , park ridge , n . j ., pp 12 - 20 . bosworth r c l ( 1952 ) heat transfer phenomena : the flow of heat in physical systems , john wiley & amp ; sons , inc ., new york , chapter ii , “ the thermal conductivity of gases ,” pp . 23 - 41 . chapman a j ( 1984 ) heat transfer , fourth edition , the macmillan company , new york . erlich h a , ed . 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