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Short Technical Reports

Rap -cyc e PCR n 1030 m n

with cycle times of 2060 s was firstemonstrate n t e ear y 1990s (1,2).

Temperature s contro e w t c rcu at ngir and samples are contained in glass

cap ary tu es. Momentary enaturat onn annea ng t mes o 0 s are ent re ydequate for PCR (3,4). Furthermore,

s ort annea ng t mes an aster trans -t ons ncrease spec c ty w t out compro-mising yield (5).

However, rap -cyc e PCR requ resccurate contro o t e samp e temper-ture, not common in most commercialnstruments w en aster protoco s arettempte . As ong as t e samp e temper-ture actually reaches the denaturationn annea ng targets, t ere s no neeor temperature o s to a ow t e samp e

temperature to catch up. Samples withing ass cap ar es eate y c rcu at ng

r ac eve accurate temperature controby matching the thermal response of thesamp e n a cap ary to a tu u ar t ermo-coup e t at mon tors t e temperature.

Recently, plastic capillaries becamevailable for use in real-time instruments

t at were or g na y es gne or rap

cycling with glass capillaries (6). Because

t e erent t erma c aracter st cs osuc tu es, t e r e ect on t erma cyc ngnd PCR amplification with rapid cycling

protoco s was nvest gate .

Instrumentation and Temperature

on tor ng

rapid-cycle, real-time PCRnstrument (L g tCyc er 1. , Roc e,

In anapo s, IN, USA) was use or astudies. Standard glass capillary tubes(20 L max mum vo ume) an or g naarouse (Roc e) were compare w t

plastic capillaries with their matchingarouse (Sens Tu e system, Osmetec ,

Roc an , MA, USA). T e samp etemperature during cycling wasmon tore y nsert ng a m n aturet ermocoup e ( TC-TT-J-403 , OmegaEngineering, Stamford, CT, USA) into10 L o water n p ast c or g ass cap -ar es so t at t e t ermocoup e t p wast the middle of the fluid column. This

t ermocoup e was use n t e or g naeve opment o rap -cyc e PCR (1,2)nd is so small (0.08 mm in diameter) that

ts e ect on t erma response s m n ma .

he capillary tubes were closed withr tosea (Ken a , Mans e , MA, USA)

nstea o t e p ast c caps supp e y t eanufacturers, in order to avoid thermo-

oup e amage. Temperature ata wascqu re w t a an e t ermometer

(Fluke Model 54, Series , Fluke,verett, WA, USA) at a rate o onceac secon an trans erre nto Exce

(Microsoft, Redmond, WA, USA) withn n rare ata rece ver (XTNDAccessrDA PC A apter, Exten e So ut ons,oise, ID, USA). Temperature accuracyt s system was w t n 0.2 w en

easure y ce water an o ng waterbaths.

ys ca u e easurements

To determine tube dimensions, glassap ar es were score an ro ena way own t e r engt . Because t e

plastic capillaries were tapered, cross-sect ons near t e ase an t p were

ta ne y gr n ng t em own on apolishing wheel (M-Prep 3, Allied High

ec Pro ucts, Ranc o Dom nguez,A, USA) w t 00-gr t paper. A CCDamera (Fire-i 400, Unibrain, San Ramon,A, USA) was use to mage t e cross-

sect ons, t e mens ons eterm ne

n pixels (QCapture Pro, QImaging,urrey, BC, Cana a) an converte tom y corre at on to a g ta ca pereasurement of the outer diameter.

ap - yc e, ea -t me

T ree temp ates were amp e yrap -cyc e PCR. A 22 p ragment o

CVRL (GenBank NC_000012.10)w t 8 GC content was amp erom uman genom c DNA us ng pr mers

5-ATCTAACTGGCAGAGTGGTC-3n -GGCCACGCTGCTTCTC-3

(7). A 277 p ragment o APOE(GenBank NC_000019.8) with 75 GContent was amplified from humanenom c DNA us ng pr mers -ACGCGGCACGGCTGTCCAAGG-3and 5 GGGCTCGCGGATGGCGCTGA-3

(8). A 300 p ragment o pBR322(GenBank J01749) with 61 GContent was amplified from a plasmid ofe DNA Too ox (9) ( n y prov ey Lonza, Roc an , ME, USA) us ng

primers 5 CGCACTTATGACTGTCTCT-3 an CCGGAAGCGAGAA

AATCAT-3 .

Plastic versus glass capillaries for rapid-cycle PCR

Oluwole Elenitoba-Johnson1 Derek David2, Niel Crews1 andCarl T. Wittwer1

1University of Utah, Salt Lake City and 2Idaho Technology, Salt Lake City, Utah, USA

BioTechniques 44:487-492 (April 2008)doi 10.2144/000112722

ap -cyc e uses ast temperature trans t ons an m n ma enaturat on an annea -

ing times of 0 s to complete 30 cycles in 10 to 30 min. The most popular platform am-

p es samp es n g ass cap ar es arrange aroun a carouse w t c rcu at ng a r or

temperature contro . ecent y, p ast c cap ary rep acements or g ass cap ar es ecame

available. We compared the performance of plastic and glass capillaries for rapid-cycle

. eat trans er n to p ast c cap ar es was s owe y t c er wa s, ower t erma

con uct v ty, an a ower sur ace area o-vo ume rat o t an g ass cap ar es. ereas t e

denaturation and annealing target temperatures were reached by samples in glass capil-

ar es, samp es n p ast c cap ar es e s ort o t ese target temperatures y .ap -cyc e was per orme on two uman genom c targets (A an A

and one plasmid (pBR322) to amplify fragments of 225300 bp in length with melting

temperatures o . . . ea -t me amp cat on ata, en -po nt me t ng curves,

an en -po nt ge ana ys s revea e strong, spec c amp cat on o samp es n g ass an

complete amplification failure in plastic. Only the APOEtarget was successfully amplified

y exten ng t e enaturat on an annea ng t mes to or s. s o ng per o was

necessary to reac target temperatures n p ast c cap ar es.

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PCR was performed in 10 L volumesw t n e t er g ass or p ast c cap ar es.Included in the reactions were 2.5 mMMg +, 50 mM Tris-HCl (pH 8.3), 500

/mL BSA, 200 M o eac eoxynu-cleotide triphosphate, 0.5M eac o twoprimers, 0.4 U of KlenTaq1 polymerase(A Pept es, St. Lou s, MO, USA),88 ng o TaqStart ant o y (C onTec ,Mountain View, CA, USA), 1 LCGreenP us (I a o Tec no ogy, Sa t La e C ty,UT, USA), an temp ate DNA. Temp ateDNA was either 1.5 104copies of humangenom c DNA or 10 cop es o p asmper react on. Rap cyc ng protoco swith programmed temperature transitionso 20 /s were use as spec e n eac

gure egen .

ro uct na ys s

After PCR, melting curves wereobtained on a high-resolution meltingnstrument (HR-1; I a o Tec no ogy) at arate of 0.3 /s between 75and 95C anda fixed LED power of 21.5 Becauset e p ast c cap ary mens ons are

erent rom t ose o g ass cap ar es,the standard HR-1 cylindrical heatingngot was rep ace w t a custom ngotmac ne out o a um num to c ose y t

the plastic capillaries. After melting, PCRpro ucts rom ot p ast c an g ass cap -laries were analyzed on 1.5 agarosegels.

RESULTS AND DISCUSSION

A p ast c cap ary system, nc u ngsubstitute capillaries and a carousel forrea -t me PCR, was eva uate or rap -cyc e PCR. Ta e 1 compares t e men-sions and thermal properties of glass andp ast c cap ar es. In contrast to stra g t

g ass cap ar es, p ast c cap ar es aretapered to allow injection molding, whicha so resu ts n greater var at on n wat c ness. Wa t c ness var at on was33 near the base and 59 near the tip oft e p ast c cap ar es, compare w t 7or g ass cap ar es. T e average men-

sions of the plastic capillaries were greatert an g ass cap ar es, nc u ng t e outer

ameter (148 ), nner ameter (143 ,and the wall thickness (165 , leading toa 23 ecrease n sur ace areato-vo umerat o. T e t erma con uct v ty o t e

plastic material used is about 7-fold less

than glass. Surface areato-volume ratio,wa t c ness, an t erma con uct v ty

suggest t at p ast c cap ar es wrespond slower to temperature changest an g ass cap ar es. Greater wat c ness var at on an cap ary taper ngin the plastic tubes also compromisesymmetry a vantages or temperatureomogene ty oun n g ass cap ar es.

The sample temperature responsew t n p ast c an g ass cap ar es, runun er ent ca rap cyc ng protoco s,is shown in Figure 1. In contrast to glasscap ar es, target temperatures are notreac e n p ast c cap ar es. In a t on,the cycle time is increased, an effect thatepen s on t e su st tute carouse , w cpparent y as a greater t erma mass.

Whenever the denaturation or annealingtemperatures are not reac e , PCR a urewou e expecte . T at s , pro uctdenaturation and primer annealing mustoccur or PCR amp cat on.

F gure 2 s ows actua PCR resu tsmplified in glass and plastic capillaries

us ng uman genom c ( CVRL anAPOE) an p asm (pBR322) DNA astemplate. Under rapid cycling conditionsw t momentary (0 s) enaturat on annnea ng t mes, rea -t me PCR w t g ass

capillaries shows strong amplification oftargets rst o serve etween cyc es

202 , w ereas p ast c cap ar es s oweither no fluorescence ( CVRL and

APOE) or a late rise in fluorescence atcyc e 3 (pBR322). H g -reso ut onmelting and end-point gel analysisdemonstrates specific products fromt e g ass cap ar es, an a sent or owmelting, small-size products from theplastic capillaries. When 5 or 10 s holding

times were added to both denaturation andnnealing segments, PCR was successfuln p ast c cap ar es or t eAPOEtarget,but not theACVRL or pBR322 target. Aslight increase in the apparent Tmof the

APOEpro uct n p ast c cap ar es mayresu t rom t e ower t erma con uct v tyf plastic and the rather rapid 0.3 /s

me t ng rate use .gure 3 s ows ow ong t ta es

samples in plastic capillaries to reachtarget enaturat on an annea ngtemperatures. T e samp e temperatureomes to within 1C of the denaturationn annea ng temperatures w t o ng

times of 1520 s.

Table 1. Dimensions and Thermal Conductivity of Glass and Plastic Capillary Tubes

Property Glass Plastic (Topas)

Base ip

Diameter (mm)

u er . . .

Inner 1.17 1.81 .53

Wall Thickness (m)

Minimum 174 235 20

Maximum

urface area/volume (mm 1 . .

Thermal conductivity (Wm-1K-1) 0.51.4 0.120.15

hermal conductivity data taken from product data sheets (Schott North America, Duryea, PA, USA, andopas Advanced Polymers, Florence, KY, USA)

F gure 1. ample temperature traces w th nlass and plastic capillaries during rapid-cycle

PCR w th momentary denaturat on and an-

neal ng.The programmed cycling protocol was5 for 0 s, 55 C for 0 s, and 75 C for 5 s. Theample reaches the target temperature for eachtage of PCR when glass capillaries are usedblack line). However, the temperature responsef samples within plastic capillaries (red line) islower. The maximum temperature achieved in

plastic capillaries is 88.7C, 6.3 C away from

the target of 95.0 C. The minimum temperaturechieved in plastic capillaries is 61.5 , 6.5way from the target of 55.0 C. The tempera-

ture range (maximumminimum) achieved byamples in plastic capillaries is only 67 of that

for samples in glass capillaries.

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490 BioTechniques www.biotechniques.com ol. 44 No. 4 2008

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Rap -cyc e PCR nstruments w tt e capa ty o 0 s enaturat on anannealing times are precisely matchedto the thermal response of the capillary.A t oug p ast c cap ar es can eused in these instruments, samples willonly reach target temperatures withenaturat on an annea ng t mes o 20 s.

Because o erent mens ons anthermal conductivity, the temperature/timecourse o t e samp e epen s strong y ont e cap ary use . It s not reasona eto expect the same PCR results in glassan p ast c cap ar es t at use t e sametemperature programm ng, part cu ar ywith rapid cycling protocols. Three PCRpro ucts o mo erate engt (22 300p) an mo erate y g GC content

(61 75 were studied here. Betterresu ts w t p ast c cap ar es may eeas er to o ta n w t ower GC contenttargets. However, the slower approach toannea ng wou e expecte to a ways

a ect spec c ty (3, ).

I concern over g ass rea agerequ res a trans er to p ast c, t e nee tore-optimize temperature/time param-eters should be expected. Rapid-cyclePCR w t momentary enaturat on annnealing will fail if capillaries of slower

thermal response are used. With 20 senaturat on an annea ng t mes, target

temperatures are reac e n p ast c cap -laries and most products can be amplified.However, t e a vantages o rap cyc ng,ot n turnaroun t me (1030 m n) an

enhanced specificity (2) are largely lost.nce re-opt m zat on s necessary w en

t e samp e vesse s c ange , trans erto more conventional thermal cyclersmay e pre erre over operat ng a rap -cyc e nstrument w t p ast c cap ar esthat results in a dynamic temperaturem smatc .

Rap -cyc e PCR w t momentarydenaturation and annealing requires aprec se matc etween t e temperature

sensor an t e samp e. One poss e

pproach is to change the temperatureresponse o t e sensor to matc a newap ary. T s as een one n t e

LightCycler 2.0 series instruments, whereifferent sensors are used for differentass cap ar es es gne to o 20 Lr 100 L. However, cyc ng spee s are

still compromised with the larger capil-ar es ecause o arger samp e vo ume,ower sur ace areato-vo ume rat o, an

increased wall thickness. Furthermore,t erma matc ng to a new cap aryrequ res ar ware (sensor) an /orsoftware modifications not available forsecon -source cap ar es. A t oug tmay e poss e to eventua y eve op aplastic substitute that allows rapid cyclingw t compara e p ys ca r g ty, wat c ness, t erma con uct v ty, anuniformity of glass capillaries, it is a tallr er to an t e reason rap cyc ng

tec n ques were eve ope w t g assapillaries (1,3,6).

any c n ca , researc , an n ustr a

pp cat ons are ocuse on t roug putrather than turnaround time and do notrequire rapid cycling. Sample procurement,transport, an samp e preparat on are

ten m t ng. Furt ermore, some otstart methods (heat activated polymerases)n econtam nat on proce ures (uracyeg ycosy ase) ncrease t e ase ne t me

required for PCR. Nevertheless, slowerPCR s n erent y ess spec c (2,3, ) anong amp cat on t mes m t t e ava -bility of expensive real-time instruments.

ap -cyc e PCR s an a vantage

w enever ast turnaroun t me an

F gure 2. Real-t me PCR, product melt ng curves, and agarose gel electrophores s of three PCRroducts. Momentary (0 s) holding times at denaturation and annealing were programmed and the

results in glass (black lines) and plastic capillaries (purple) compared. Samples in plastic capillaries werelso amplified with 5 s (yellow) and 10 s (blue) holding times at denaturation and annealing tempera-

tures. A 225 bp CVRL1gene fragment of 68 GC content and T of 93.1 C is shown in (A), amplifiedfter an initial denaturation of 96 for 10 s by 40 cycles of 68 for 0 s, 75 C for 5 s, and 94 C fors. (B) shows a 279 bp fragment of POEwith 75 GC content and a Tmof 90.3C amplified under

the same conditions asACVRL1except that 7 DMSO was present. (C) shows a 300 bp fragment of

pBR322 with 61 GC content and a Tmof 92.9 , amplified under the same conditions asACVRL1 -ept with an annealing temperature of 57 . The molecular size marker (BioMarker Low, BioVentures,Murfreesboro, TN, USA) is on the right lane of each gel.

Figure 3. Denaturation and annealing holdingtimes required for the sample to approach targettemperatures in plastic capillaries. The instru-ment was programmed for denaturation at 95 C,nnealing at 55 , and extension at 72 C for 5 s.

The programmed holding times at denaturationnd annealing target temperatures varied from

0 to 20 s. The high and low sample temperatures

reached during cycling are plotted against theholding times at denaturation and annealing.

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high specificity are desirable. Clinicalscenar os nc u e acter a spec at on(10), ant ot c res stance eterm nat on(11), an p armacogenet c test ng ortherapeutic dosing (12). Research applica-tions include DNA engineering requiringmany sequent a PCRs (13). Rap -cyc ePCR s est per orme on sma vo umesamp es an s a popu ar tec n quein microdevices (14,15). With largervolumes commonly used in standardequipment, rapid PCR in 1030 min isst poss e t e samp e temperature sccurate y contro e . Heat ng samp esn g ass cap ar es w t c rcu at ng a r

provides a unique solution for rapid-cyclePCR. However, use of plastic capillariesn systems matc e to g ass cap ar es

requ res emp r ca opt m zat on at s owerspee s w t comprom ses n turnarountime, temperature homogeneity, and PCRspecificity.

ACKNOWLEDGMENTS

T is wor was supporte y NationaInstitutes o Hea t (NIH) grants (nos.GM7 an GM7 ).

Aspects o rapi -cyc e PCR, rea -timePCR, an me ting ana ysis are icenserom t e University o Uta (Sa t La e

City) to I a o Tec no ogy an rom I a oTechnology to Roche Applied Science.C.T.W. holds equity interest in Idaho

Tec no ogy. D.D. is an emp oyee o I a oTec no ogy.

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4. ttwer, .T. and M.G. Herrmann. 1999.apid thermal cycling and PCR kinetics,. 211-229. In Innis, M., D. Gelfand, and. Sninsky (Eds.), PCR Methods Manual.

cademic Press, San Diego, CA.

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. Highsmith, .E., Jr., Q. Jin, A.J. ataraj,J.M. Connor, V.D. Burland, W.R.Baubon s, F.P. urt s, . usukawa, andM.M. arner.1999. Use of a DNA toolboxfor the characterization of mutation scan-

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12. Carlqu st, J.F., J.T. McK nney, Z.P.

N cholas, J.L. Clark, S.F. Kahn, B.D. Horne,J.B. Muhleste n, H.T. May, et al. 2007. Rapidmelting curve analysis for genetic variants thatunderlie inter-individual variability in stablewarfarin dosing. J. Thromb. Thrombolysis.[Epub ahead of print, July 29, 2007].

13. Brown, .A., .J. ay, and C.T. W ttwer.1998. Rapid cycle amplification for construc-tion of competitive templates, p. 57-70. nHorton, R.M. and R.C. Tait (Eds.), GeneticEngineering with PCR. Horizon ScientificPress, Norfolk, VA.

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.15. Zhang, C. and D. X ng.2007. Miniaturized

PCR chips for nucleic acid amplification and

analysis: latest advances and future trends.Nucleic Acids Res. :4 -4 .

Received 13 October 2007; accepted

December 2007.

Address correspondence to Carl T. Wittwer,

Department of Pathology. 50 North

Medical Drive, University of Utah, Salt

a e ty, , , . e-ma : car .

w ttwer pat .uta .e u

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