Nucleic Acids Research, Vol. 19, No. 1 99

Post-PCR sterilization: a method to control carryovercontamination for the polymerase chain reaction

George D.Cimino*, Kenneth C.Metchette, John W.Tessman, John E.Hearst1 andStephen T.lsaacsHRI Research, Inc., 2315 Fifth Street, Berkeley, CA 94710 and 'Department of Chemistry, Universityof California, Berkeley, CA 94720, USA

Received September 12, 1990; Revised and Accepted November 30, 1990

ABSTRACT

We describe a photochemical procedure for thesterilization of polynucleotides that are created by thePolymerase Chain Reaction (PCR). The procedure isbased upon the blockage of Taq DNA polymerase whenit encounters a photochemically modified base in apolynucleotide strand. We have discovered reagentsthat can be added to a PCR reaction mixture prior toamplification and tolerate the thermal cycles of PCR,are photoactivated after amplification, and damage aPCR strand in a manner that, should the damagedstrand be carried over into a new reaction vessel,prevent it from functioning as a template for the PCR.These reagents, which are isopsoralen derivatives thatform cyclobutane adducts with pyrimidine bases, areshown to stop Taq polymerase under conditionsappropriate for the PCR process. We show thateffective sterilization of PCR products requires the useof these reagents at concentrations that are tailored tothe length and sequence of the PCR product and thelevel of amplification of the PCR protocol.

INTRODUCTION

Nucleic acid probe technology has been an essential tool formolecular biology since its inception 30 years ago. During thelast decade, quantum developments in this field resulted fromthe introduction of automated DNA synthesizers. Short, syntheticoligonucleotides provided new tools for the advancement ofcloning techniques, the creation of allele-specific hybridizationprotocols and, most importantly, template-dependent polymeraseextension reactions were developed into nucleic acid amplificationprotocols by using the oligonucleotides as specific primers (1-4).The first of these procedures, the Polymerase Chain Reaction(PCR) (5,6), is now an established technique worldwide. Onehundred to several thousand bases of a few molecules from a

single target sequence in the presence of complex genomic nucleicacid sequences are routinely amplified to as many as 1012-1013molecules in just a few hours. This technique has revolutionizedthe way molecular biologists conduct experiments, and has foundapplication in a variety of different fields.

Contamination by 'Amplicon' Carry-OverThe widespread use of the PCR has come about because of itsexquisite specificity and sensitivity. These features, however,have one drawback: the synthesis of specific PCR products thatarise through the amplification of previously amplified sequences

('amplicons') which are inadvertently carried over into a

subsequent PCR reaction (7-9). These carryover amplicons cancontaminate reagents or sample specimens, causing systematicerrors. Alternatively, individual reaction vessels can becontaminated during the preparative procedures and result insporadic false positive PCR signals. The carryover problem ismost acute in environments where the same amplification is runtime and time again (e.g. a clinical diagnostic lab). Extreme care

is essential when handling amplified samples so that carryover

is minimized. The recommended sample handling conditions are

similar to those used by microbiologists for the handling andgrowth of microorganisms(10,11). In fact, the problem isnumerically analogous since similar amplifications occur whenbacteria or viruses are grown in large volumes. Biologicalorganisms are, however, effectively sterilized by numerous

techniques that either thermally or chemically denaturemacromolecules. Simple denaturation is not adequate forsterilization of PCR products since a single strand of a nucleicacid is readily amplified and is also chemically resistant to thetemperatures encountered in laboratory sterilizers. Effectivesterilization of amplicons requires chemical, photochemical or

enzymatic modification of the amplicons that prevent them fromacting as templates for polymerase extension reactions.

General Considerations for the Sterilization of AmpliconsCreated by the PCR ProcedureFrom a temporal point of view, there are two places in a PCR-based diagnostic assay where sterilization can be implemented.When performed just before amplification ('pre-PCRSterilization'), carryover molecules present in the reaction mixwill be rendered sterilized. When performed after amplification('post-PCR Sterilization'), all nucleic acids, including PCRproducts, will be sterilized. These two sterilization modes are

distinguishable in that they impose specific and different

* To whom correspondence should be addressed

100 Nucleic Acids Research, Vol. 19, No. I

requirements upon the nature of a chemical modification processand upon the reagents present during the modification prcre.With the pre-PCR sterilization mode, PCR r sUscptible

to the chemical modification process must be left out and addedback to the reaction mix before amplification can take plce.These reagents most likely include Taq polymerase, the true targetnucleic acid, and the primer oligonucleotides. Sporadic PCRerrors due to amplicon carryover sfill exist with this mode sincecarryover molecules can be introduced during the addition of theother reaction components. Recently, a direct, shortwave UVirradiation procedure has been described as a pre-amplificationsterilization process (12). This technique can sterilize some PCRreagents, but the process is inadequate for the entire complexityof carryover problems (13). An enzymatic approach thatdistinguishes amplicon from true target and primeroligonucleotides would be advantageous as a pre-PCR ilizationtechnique if the enzyme is also compatible with the PCR.With post-PCR sterilization, all of the PCR reagents can be

present since polymerase activity, primer integrity, and true targetintegrity are not of concern following amplification. Sterilizationin this mode can potentially obviate both the systerdlatic andsporadic errors associated with the carryover problem if all ofthe amplicons are sterilized before the raction tube is openedand exposed to the environment. With this mode, however, thechemically modified PCR strands must sfill be detectable. Thisusually implies that either a) the modified PCR amplicons becapable of specific and efficient hybridization to anoligonucleotide probe which is complementary to an internalsequence within one of the PCR strands, or b) that the modifiedamplicons be detectable as an oligomucleotide product of a specificlength when analyzed by gel electrophoresis.A post-PCR sterilization process that employs a phoochemical

reagent should have additional properties to be used convenientdywith the PCR process. First, the non-activated reagent must notinterfere with primer annealing or Taq polymerse acivity, andmust be thermally stable to the temperatures enountered withthe PCR technique. This permits the reagent to be added priorto amplification. Second, the ideal reagent would be activatedby wavelengths between 300 and 400 nm since the polypropylenetubes used for PCR reactions are transmissible to thesewavelengths, direct UV damage to the amplicons is minimized,and PCR samples can be handled on the bench with normalfluorescent or incandescent lighting. Finally, the oochemicalmodification of the amplicons must prevent the modified ampliconfrom acting as a template for the PCR.

This last criteria assumes that an amplicon with a singlemodification is prohibited from acting as a PCR templatemolecule. Even with this assumption, the effectiveness of anynucleic acid sterilization process will also be dictated by themagnitude of the carryover problem, the extent of theamplification, and the average degree of modification of theamplicons. The population of carryover amplicons must bemodified to a sufficient average extent per molcle so thatstatistically there is a small likelihood tat some molecules arewithout any modifications, and therefore non-sterilized. Someof these non-sterilized carryover molecules, however, can betolerated if their total number is less thn the minimum detectionlimit of the PCR procedure.

Table I illustrates the relationship between detection sensitivityand PCR amplification at different numbers of initil targetmolecules. The amplification factor that is observed with the PCRtechnique is related to the number (n) of cycles of the PCR thathave occurred and the efficiency of replication at each cycle (E),

which in turn is a function of the priming and extensionefficiencies during each cycle. Ampliflcuiou follows the form(1 + E)n until high concentrations (approx. 10-,8 M or greatet)of PCR product are made. PCR prodc elly reaciXaplateau concentration. Also shown in TatkI is a signal (CPM)for a hybridization assay that is used to detect Xt presence ofPCR product. Using 100 CPM as a detection limit, the data ofTable I show that detection of a single copy gene from 1 g -ofgenomic DNA requires only 20 cycles ofaflation.Uthese conditions, several thousand carryover moleI es can escapedetection. By contrast, the identification-of 10 taget moleculesrequires 30 cycles of amplification. Under e conditions, jusa few molecules of carryover will generate a false positive PCRsignal.We describe here a photochemical process that destroys the

template activity of PCR amplicons yet permits the modifiedamplicon to be probed in hybridization reactions. The processcan be designed to ensure that carryover as high as 1012molecules are incapable of being amplified by the PCR procedure.

MATEIRIALS AND METHODSMaterialsMaterials and light soures were ob d scified in thaccompaying manuscript (14). 5-MUIP eyn and3H-5-MIP were obtained from HRI Associates, Inc. (Berkeley,CA).

PCR AmplificationsAn amplicon 115 bases in length wasgeaed by using primevaSK38 and SK39 according to publisd rdues (15) witieither 1 pg of genomic DNA from an HIV-1 poitive patient asthe initial target or known copy numbers of the -1Smer amnicongenerated in a previous PCR reaction. A 50Ybas pair amplionwas also generated for comparative purposcording to tproocol described in the GeneAmp DNA aKit (#N801-0055, Perkin-Elmer/Cetus) b ting piPCR1I/PCRO2 with the control bacterioplg tlate or withPCk products generated in a previous PCR reaction with thisprimer/target system.

Photoaddition ReactionsIrradiations. All irradiations were performed wi either a device(PII) described previously (16) or the HRI-1OOdevice. The PTIdevice exposes one sample at a time and has no emission below320 nm. By contrast, the HRI-100 device is 'designed for thesimultaneous irradiation of multiple Eppetuf tubes that areroutinely used for PCR amplifications. This device consists ofsix- fluorescent lamps (Black Light type FS&TBL) arrangedparallel to each other. From a side view, the lamps form a U-shaped trough. A double-walled, UV-htasparent plastic holderrests in the trough which supports up to 48 Eppendorf tubes (0.5ml). With this arrangement, the sample tubes are exposed toapproximately 20 mW/cm2 of 300-400 tnm light. StandardEppendorf tubes have an average of 10% -trasmission for thiswavelength region. During irradiations, sampl werem intainedat room temperature by passing watertugh the doubled-walledsamnple holder. We have observed that some amnplicons are moreefficiently sterilized when exposed to light at lower temperatures(4°C, data not shown).

Photoaddition to Genomic DNA. 3H-4f-AMDMIP (2.2 X105CPM/pg), 3H-AMIP (3.1 x 105 CPM/pg) or 3H-5-MIP

Nucleic Acids Research, Vol. 19, No. 1 101

Table I: PCR amplification as a function of initial target number and cycle number.

102 103 104 105 3x105*

AmplificationCycle FactorLL.-Ur 2.2 x 105 2.2 x 106 2.2 x 107 2.2 x 108 2.2 x 109 2.2 x 1010 6.6 x 1010

20 2.20 x 105 3.6 x 10- 15 3.6 x 10- 14 3.6 x 10- 13 3.6 x 10- 1 2 3.6 x 10- II 3.6 x 10-10 1.0 x io-9

0 0 2.4 24 240 2,400 7,100.............. -1-111-1

4.8 x 4.8 x 108 4.8 x 109 4.8 x 1010 4.8 x I 0 :::':A--

9106 7.9 10-14 7.9 10-25 7.9 x 10- II 7.9 x 10-10 7.94.78 x 7.9 x 10- 12 8

0 5 5 2 520 5,200 5.2 x I04

.............................

..

... .. ...........1081.0 x 1.0 x 109 1.0 x 10, 0 1.0 lo, ::::::I :4-:il...................A .......................

-.-+.......+.......-...................

1 08 9 ...: ............30 1.03 1.7 x 10-12 1.7 x 10- I 1 1.7 x 10-10 1.7 x 10- ...............

......... ......................04 .............1,100 1 .1 I t .............................

......

....................

......................... ..........................................

................ ..................................... .............................. ................... ............................

...1092.2 x 2.2 x 1010 2.2 x 101 I.... ............ .......-..............................................................................................

V:+""","","",","""""""""""""""", '' ................. ....... .. ... .... .......35 2.24 x 109 3.6 x 10- II 3.6 x 10-10 3.6 X 10-9.........................................-..............

..............................*..........................

......................-..................................................

......... ....... .........

240 2 ,400 2 .4 104 ................... .........

.... ....

...............

... ... ........

............................

.. .. ............. .....

IT -1010 4.9 101 I .... .......4 .9 x ::::A ".: ............................. .............................:::X,::::Io +:" """"

* I ug genomic D

PCR product molccules

PCR Product ( M/1)

Hybridization Signal (CPMs)

)NA

Each block in this table contains three sets of data: (1) The total number of PCR product molecules present after the initial number of target strands are amplifiedfor the number of cycles shown. These numbers of amplicons were calculated using an amplification factor of (1.85)f. We have experimentally confirmed the valueof this factor for the 1 l5mer amplicon generated with the primers SK38 and SK39. (2) The concentration of PCR product when the amplification takes place ina 100 il volume. The gray area indicates conditions that give rise to PCR products that are in excess of 10-8 M. In these regions, the PCR plateau effect willbe acting on PCR product synthesis. And (3), a hybridization signal that is used to detect the presence of PCR product. This signal is calculated on the basis ofhaving a 10% hybridization efficiency of a 32p labelled probe (3000 Ci/mM) to a 20 I1 aliquot of the 100 pa PCR reaction mix.

(2.1 x 105 CPM/yg) at the concentrations described in the textwere mixed with 100 ,ug of calf thymus DNA (Sigma ChemicalCo.) in 200 Al of 1 x TE buffer, pH=7.0. 30 td aliquots of thesesamples were irradiated at room temperature with the HRI-100device for the time periods indicated. Photobinding levels were

determined as described in the accompanying manuscript (14).

Photoaddition to Synthetic 115mer. Photobinding of3H-4'-AMDMIP to the double stranded 115-mer(HRI-46/HRI-47) was determined as described in theaccompanying manuscript (14).

Sterilization Assays115mer Sterilization. 1 Ag of genomic DNA from an HIV-1positive patient was sufficiently amplified with primersSK38/SK39 to produce a plateau PCR reaction. Identical aliquotsof this PCR mixture containing approximately 106 copies of the1 i5mer amplicon in 0.5 ml Eppendorf tubes were adjusted to100 /.g/ml 4'-AMDMIP in a total volume of 10 Il containing

1 x Taq buffer (50 mM KCl, 2.5 mM MgCl2, 10 mM Tris, pH8.5, 200 pg/ml gelatin). Half of these samples were irradiatedfor 15 minutes at room temperature with the PTI device. Theirradiated samples, as well as control samples, were adjusted to20 1d by the addition of new PCR reagents. To determine thetemplate activity of these amplicons, the PCR reaction mixturescontaining 1 x Taq buffer, 200 AM of each dNTP, 5 ,uCi ofa-32P-CTP (3000 Ci/mM, New England Nuclear), 0.5 unit ofTaq DNA polymerase (Cetus/Perkin Elmer) and 0.5 ,^M of eachSK38 and SK39 primers were then reamplified. After adding 5

tl of a dye solution, 10 Itl of each of the reamplified mixtureswas run on a 12% polyacrylamide/8M urea gel, and analyzedby autoradiography. By comparing with known copy numbersof a synthetic 1lSmer oligonucleotide, the alpha incorporationassay will detect at least 100 copies of template 1 iSmer after

30 cycles of amplification.

115mer and SOrmer Comparison. To compare the sterilizationefficiency of these two different amplicons, equivalent copy

INITIAL COPY NUMBER 10

102 Nucleic Acids Research, Vol. 19, No. I

numbers of each amplicon were initially prepared in the followingmanner: A 30 cycle PCR reaction was first carried out for eachsystem with the appropriate primers and starting templatemolecules. Aliquots (approximately 105-106 target copies) ofeach of these reactions were transferred to a second set of PCRreactions. The second sets of PCR amplifications were carriedout in the presence of a1-32P-dCTP (low specific activity), againfor 30 cycles. Aliquots of these reactions were run on a 3%NuSieve/1 % agarose gel, and then cut and counted by liquidscintillation counting in order to determine the exactconcentrations of each of the two amplicon products in the second

set ofPCR reaction tubes. The concentations of both the 1 5merand the 500mer were then adjusted to 1x 10-8 M by theaddition of additional Taq buffer. These solutions were made 100pg/ml in 4'-AMDMIP. Both samples were subsequently dividedinto two portions, one part being irradiated for 15 minutes atroom temperature with the HRI-l00 device and the other partkept in room lights. Serial dilutions of the irradiated and theunirradiated amplicons were carried over into new tubes

(20 ul total volume) containig new PCR reagents and highspecific activity a-32P-dCTP, and then reamplified for another30 cycles of PCR. 10 yl aliquots of these samples were analyzedon 12% polyacrylamide/8M urea gels.

Template Dependent Extension Experiments with Taq DNAPolymeraseSynthesis of 71mer-MA Templates. Three templateoligonucleotides (71 mer-MA's) containing site-specificisopsoralen monoadducts (MA) were constructed. The 71mnersequence (5'-ATC CTG GGA TTA AAT AAA ATA GTAAGA ATG TAT AGC CCT ACC AGC ATT CTG GACATA AGA CAA GGA CCA AA-3') is a subsequence of the1 l5mer amplicon. SK39 is complementary to the last 28 basesof the sequence above.

Preparation of 71mers which contain site-specific monoadductsinvolved 1) preparation of different l5mer monoadductedoligonucleotides from the same unmodified l5mer and 2) ligationof the different i5mer monoadducted oligonucleotides to the same56mer extender oligonucleotide using a 25mer oligonucleotideas a splint. Isopsoralen monoadducted i5mers (1Smer-MA's)were made fiom reaction mixtues contining 100 yg ofthe l5mer(5'-ATC CTG GGA TTA AAT-3') and 65 isg of acomplementary lOmer (5'-ATT TAA TCC C-3') in 185 yd ofTE buffer (10 mM Tris, pH= 7.5, 1.0 mM EDTA) and 0.1 MNaCl. An isopsoralen derivative (either 4'-AMDMIP, AMIP,or 3H-5-MIP) was added to separate reaction mixtures inEppendorf tubes at 20-30 itg/ml. These 3 solutions wereirradiated with the PTI device for 15 minutes at 150C. Themixtures were then extracted with CHCl3 (4 X) and theoligonucleotides were recovered by ethanol precipitation (2 X).Unmodified l5mer was separated from 15mer-MA's byfractionation on HPLC (C18 reverse phase) using a 0. IMNH4OAc/Acetonitrile gradient. Fractions containing 15mer-MA's were identified by 5' end labelling aliquots of the HPLCfractions and analyzing these 32p labelled oligonuCleotides byautoradiography after separation on a sequencing gel. Fractionswith l5mer isopsoralen monoadducts were further purified byethanol precipitation. 71mer-MA's were constnruted with astandard ligation reaction mixtue (50 Al total volume) coainingone of the l5mer-MA's (4x 0-4 M), a 5' phosphorylated56mer (5'-pAAA ATA GTA AGA ATG TAT AGC CCT ACCAGC ATT CTG GAC ATA AGA CAA GGA CCA AA-3';5 X l0-4 M) and a 26mer complementary oligonucleotide

(5'-CAT TCT TAC TAT TTT ATT TAA TCC C-3'; 5 x 10-4M). After 2 hours of ligation, the reaction, mixtures were appliedto a 12% polyacrylamide/8 M urea gel. The 71mer-MA bandson the gel were identified, cut, eluted, and finally purified byethanol precipitation.

Taq Extension Assay. For the extension experiments, SK39 was5' end labelled with 32P by a kinase reaction and purified on asequencing gel (17). 32P-SK39 (1 x 10-8 M) was hybridized toeach of the 71mers (lx l0-9 M) in a 20 IL reaction mixconsisting of 1 xTaq buffer and 200 gAM each of NTP's. Thesamples were initially heated to 95°C for 5 minutes, thenincubated at 55°C for 3 minutes. The extension reactions wereinitiated by the addition of Taq polymerase (Q.05 units/4l) andterminated by the addition of 0.1 d of 0.5 M EDTA. Extensionproducts (10 tld) were analyzed on a 12% polyacrylamide, 8 Murea gel.

RESULTS AND DISCUSSIONPhotochemical Sterilization with FurocoumarinsFurocoumarins are a class of planar, tricyclic compounds thatare known to intercalate between base pairs of nucleic acids (18).These compounds contain two reactive double bonds which, whenexcited by 320 nm-400 nm light, react with the 5,6 double bondof pyrimidines to form cyclobutane rings. Both monoadducts andcrosslinks are formed with psoralens (linear furocoumarins) whenthey react with DNA. Psoralen crosslinks are known to blockpolymerase extension reactions presumably by preventing strandseparation of complementary strands ofDNA (19). Monoadductsof certain psoralen derivatives have also been reported to blockextension reactions with some polymerases, but blockage bymonoadducts is not observed in all cases (19-22). This may bedue to the fact that, although psoralen crosslinks induce adeformation of a DNA helix at the adduct site (23), psoralenmonoadducts do not produce similar alterations ofDNA helices(24). Thermodynamic studies agree with the structural studiesand show that the presence of a psoralen monoadduct on anoligonucleotide does not substantially effect the hybridizationstability of the oligonucleotide (25). Isopsoralens (angularfurocoumarins) have been presumed to be similar to psoralensin their reactivity with nucleic acids except that they form onlymonoadducts because of the relative orientation of the reactivebonds of these molecules. Oligonucleotides modified by someisopsoralen derivatives are known to retain hybridizationcapabilities (26).We have evaluated derivatives of both psoralens and

isopsoralens as photochemical reagents in a post amplificationmode for PCR sterilization. While derivatives of both classes ofcompounds are capable of sterilizing PCR amplicons, theisopsoralen modified PCR product oligonucleotides can be probedby hybridization and are therefore favored. Modificationrequirements for effective sterilization of PCR products withisopsoralens are described below. Similar requirements apply topsoralen sterilization.4'-AMDMIP (27) was evaluated for its ability to act as a

photochemical sterilization agent for PCR amplicons. Thesterilization protocol is outlined in Figure la. An amplicon 115bases in length was generated by performing PCR on genomicDNA under conditions that yield amplicon at plateauconcentrations. Several identical samples, each coinaining 106copies of the amplicon, were aliquoted from the plateauamplification mix. 4'-AMDMIP was added to each sample. Half

Nucleic Acids Research, Vol. 19, No. 1 103

Prepare patientsample DNA

PCR Taqreagents I1 polymerase

M

UW Mie components

1 1

V

20 25

cycles cycles

I

I PCR amplification

14 1~II

lW

Aliquot into new tubes at10 6 copies per tube

lo-------- Add AMDMA(A100 pgml)

vg ll- rTradicae Noe15 mins irradiation

{ Add rnew PCR reagerits {

30cycles Reamplily with a- 32 P*dCTP

'g20

cycles

20 cycles 25 cycles 30 cycles

+ + - + t1

115 -mer

Analysis by Denaturing P'AGE

20 25 30_ r

+ + + - + + + - + + +

hY ---+ --

Lane 2 3 4 5 6 7 8 9 10 11 12

:::-

Figure 1. (A) Protocol for evaluation of 4'-AMDMIP as a photochemicalsterilization reagent for PCR amplicons. (B) Autoradiograph of the reamplificationof 106 copies of 4'-AMDMIP sterilized 1 l5mer amplicon. The samples in lanes1-4, 5-8, and 9-12 were reamplified for 20, 25, and 30 cycles of the PCR,respectively. Lanes 1, 5, and 9 are negative controls. Lanes 2, 6, and 10 indicatethe level of reamplification of unsterilized amplicon. Lanes 3, 7 and 10 are controlreactions which show that 4'-AMDMIP in a PCR reaction mix does not affectamplification yield. Lanes 4, 8 and 12 show the reamplification products obtainedwith sterilized 1 l5mer templates.

of the samples were irradiated while the other half received noirradiation. All of the samples were then mixed with fresh PCRreagents, reamplified, and finally sterilization was evaluated bygel electrophoresis and autoradiography. The reamplificationresults are shown in Figure lb. The control reactions that haveno carryover (lanes 1, 5, and 9) show no amplified product. Thecontrol reactions that contain 106 molecules of carryover without4'-AMDMIP (lanes 2, 6, and 10) generate reamplificationproducts. Direct irradiation of similar samples without4'-AMDMIP present has no effect on reamplification (data notshown). The control reactions that contain carryover with4'-AMDMIP but without light exposure generate reamplificationproducts that are equivalent in magnitude as those reamplifiedin the absence of 4'-AMDMIP. 4'-AMDMIP, as a post PCRsterilization reagent, did not affect PCR amplification efficiency.And finally, the reactions that received carryover, 4'-AMDMIPand irradiation show isopsoralen sterilization of PCR ampliconsto a degree that is cycle dependent. With 20 cycles, sterilizationappears to be complete. With 25 cycles, reamplification productis visible (lane 8), albeit reduced relative to controls (lanes 6 and7). And with 30 cycles, no significant sterilization is observed;PCR reamplification product (lane 12) is approximately the samerelative to controls (lanes 10 and 11).

This data illustrates the interplay between sterilizationsensitivity and the amplification factor. At 20 cycles of PCRamplification, sterilization appears to be completely effective. If100 CPM is taken to be the threshold for seeing a band on theautoradiograph, then Table 1 shows that the sterilization protocolof this example with 4'-AMDMIP left less than I04 ampliconsthat were capable of being replicated by the PCR procedure. At25 cycles of PCR, a very measurable band is observed, suggestingthat at least 103 amplicons retained replicating capabilities. At30 cycles of PCR it is difficult to distinguish the control signalfrom the signal obtained with the sterilized sample. This isconsistent with both the control sample and the 4'-AMDMIPtreated sample reaching the plateau region of the PCRamplification process.

There are at least two factors that would make the sterilizationprotocol just described inadequate for treating the 106 copies ofcarryover amplicons. First, the sterilization process will beincomplete if Taq polymerase is capable of polymerizing throughan isopsoralen adduct on an amplicon. To evaluate the extentthat Taq polymerase reads through isopsoralen monoadducts, weconstructed three modified oligonucleotides, each of whichcontained a different isopsoralen at specific positions near the5' ends of the oligonucleotides. These oligonucleotides were thenhybridized to a labelled primer (SK39 in Figure 2a). This primerwas extended with Taq polymerase and the length of thepolymerase products were determined. The isopsoralen modifiedoligonucleotide templates were constructed by assuming thatisopsoralens, like psoralens, would have as a preferentiallyreactive site a 5' TpA-3' sequence in duplex DNA (28,29). Webegan with two short complementary oligonucleotides that containa single TpA site within their complementary sequence region.These oligonucleotides were reacted with either 5-MIP,4'-AMDMIP or AMIP under conditions where the twooligonucleotides would be hybridized. 15mer monoadductedoligonucleotides were purified from non-modifiedoligonucleotides and then ligated at their 3' ends to anotheroligonucleotide to generate the monoadducted templateoligonucleotides (71mer-MA's).

Extension products generated with the 71mer templates areshown in Figure 2b. The 71mer-MA template that was made with

A

B# PCR Cycles

TargetAMDMIP

104 Nucleic Acids Research, Vol. 19, No. I

0* --*.--. '- 3'

5 3

3' i~3.~~~~~.SK39

(Labelled. *)

*3' Taq Polymerase

z>5' dNTP's

B I emplate C

ExtensionConditions (1)

Lane 1

71 mer MA C(5-MIP)

1)\ (2) (3) (2} (1)

2 3 4 5 6

71 mer MA(AM IP)

(2) (3) 3

7 8 9

.;:r .- X,-~r hN; v

'1 i13

l .: 3

Primer -

Figure 2. (A) Protocol for the analysis of Taq polymerase exnsiou reacto with isopsoralen monoadducted oligonucleotide templates. (B) Autoradiograph of the

extension products produced with Taq DNA polymerase and template molecules containing isopsoralen monoadducts. The extension of SKI9 by Taq DNA polymeraseon the 71mer templates was evaluated under 3 sets of extension conditions: Condition (1) corresponds to adding Taq polymerase at 55°C to a reaton tube andpermitting the extension to proceed for 1 minute at 550C. The addition of EDTA terminated the reaction. Condition (2) corresponds to the same 1 minute extensionat 550C, followed by transferring the tube to another bath at 72°C and allowing the extension reaction to proceed for another minute. Again, EDTA was used toterminate the reaction. Condition (3) corresponds to condition (2) folowed by a strand separation step (30 sec at 95°C). The reaction tube was then brought to55°C again for an additional 1 minute, followed by 72°C for 1 minute and finally 95C for 30 sec. This thermal profile was repeated for a total of ten cycles,and then EDTA was added to terminate Taq polymerase activity. Lanes 1, 5, 9, and 13 show the extension products that are generated with a synthetic 71mertemplate control oligonucleotide. Lanes 2-4, 6-8, and 10-12 show the extension products that are obtained with the template molecules that were constructedwith l5mer-MA's containing 5-MIP, AMIP, and 4'-AMDMIP, respectively.

5-MIP produced a single truncated extension product when the

extension reaction was run for 1 minute at 55°C. Similar resultswere obtained when the extension protocol included a second1 minute extension at 72°C. When the extension protocol was

cycled 10 times to mimic PCR, evidence of a small amount ofa flail length extension product was visible. The 71mr-MA targetcontaining 4'-AMDMIP monoadducts yielded similar results forthe 3 extension conditions. However, multiple stops were

A

5'

71 ner - MA

MA Li

3'

cte

'-

'*",v-5'

-lq-mjm.....lipff-"

Nucleic Acids Research, Vol. 19, No. 1 105

U)

a-

a)ftA

cU9.6 ug/mI

o > > * 45.2 ug/mI

CD 100-- 95.9 ug/mI

-~359 ug/mI

0 1 0 20 30 40

Irradiation Time (Minutes)

Figure 3. Photoaddition of 4'-AMDMIP to genomic DNA.

observed with this isopsoralen and the predominant stop was a

position entirely different from that produced with the 5-MIPcontaining template molecule. The 71mer-MA target containingAMIP monoadducts produced 3 truncated products upon

extension with Taq polymerase. Again, the 10 cycle extensionproduct produce some full length product.

These extension experiments show that all 3 isopsoralen adductsare effective blocks for Taq polymerase. While some full lengthproduct did occur with each of the isopsoralen templatemolecules, our data is insufficient to conclude that truereadthrough exists. We have quantitated the readthrough withanother set of experiments using a 5-MIP modified template. Forthese experiments, the HPLC purified l5mer-MA oligonucleotidewas further purified by PAGE prior to the ligation reaction toform the 71mer-MA. With this template molecule, the full lengthextension product accumulates at 0.2 % per cycle relative to thetruncated product band. This data implies that our 71mer-MAwas either 99.8% pure or that readthrough occurs at a 0.2% rate.This issue is not resolved at present because we cannot determinethat the initial 1Smer-MA was greater than 99.8% pure.

The extension experiments also suggest that the three differentisopsoralens have different preferential binding sites on DNAmolecules. The stop with the 5-MIP template appears to be atabout the position of the TpA sequence in the l5mer that was

used to create the monoadduct. The longest extension productswith the AMIP and ADMIP templates indicate that theseisopsoralens probably reacted with the initial iSmer outside theregion of the lOmer/1Smer hybrid interaction. The sequence ofthe l5mer does not indicate any obvious secondary structure inthis oligonucleotide, suggesting that the two positively chargedphotochemical reagents, 4'-AMDMIP and AMIP, may beinteracting with single-stranded regions of DNA. This is a

desirable feature for a PCR sterilization reagent and has beenconfirmed elsewhere (14).The extension experiments performed with 4'-AMDMIP

monoadducted template molecules show that it is unlikely thatthe data of Figure lb arose from bypass synthesis by Taqpolymerase during reamplification. Furthermore, while 5-MIPadducts are effective stops for Taq polymerase, this compoundis inadequate as a photochemical sterilization reagent (see below;data not shown). A more plausible explanation for the inadequatesterilization is that the protocol used to generate the data in Figurelb provided incomplete modification: some of the ampliconmolecules were not modified and therefore, not sterilized.

Photochemical sterilization with isopsoralens is fundamentallya statistical process. This process is characterized by measuringthe average number (a) of adducts per amplicon strand. If theaddition reaction is governed by Poisson statistics, the fractionof molecules with no modifications in a large population ofamplicons that have an average of a modifications is given byfa(0) = e-a. As in the example of Figure 1, if the carryoverevent consists of 106 amplicons, 2.5 x 103 non-sterilizedtemplate molecules will be present when the modification protocolgenerates an average of only 6 effective adducts per strand ofPCR amplicon. Effective adducts are defined as adducts whichoccur in the segment of a target molecule bounded by the primersequences. For the 1iSmer of Figure 1, this level of effectiveadducts occurs when there is an average strand modificationdensity of 1 adduct per 9.3 bases. The data of Figure lb canbe explained if the sterilization protocol yielded a similarmodification density. We have measured the binding of tritiated4'-AMDMIP to synthetic strands of the 1l5mer ampliconproduct. Under identical irradiation conditions (with the PTIdevice) as those for the sterilization experiment, a modificationdensity of 1 adduct per 8.4 bases was measured. This data isconsistent with sterilization efficiency being determined by thestatistical nature of the photochemical modification procedure.

Optimization of Nucleic Acid Sterilization with IsopsoralensIsopsoralens undergo photochemical degradation concomitantwith photochemical addition to polynucleotides. The ratio ofdegradation to binding is a function of the isopsoralen derivativeand the irradiation wavelengths. These competing reactions resultin a plateau level of addition of these compounds to nucleic acidsthat is dependent on the initial concentration of the photochemicalreagent during the activation process and on the wavelengths usedto excite the molecules. When the synthetic 1 l5mer was irradiatedfor 15 minutes from a UV source that enhanced binding(HRI-100), a modification density of 1 adduct per 5 bases wasobtained with 4'-AMDMIP at 100 ug/ml. This high level ofmodification is unique to this short amplicon which contains 70%AT in the sequence region bounded by the primers. Ampliconsof this short length display sequence dependent modificationdensities. Figure 3 illustrates the modification densities that areachieved with 4'-AMDMIP when it is reacted with long, complexgenomic DNA. Modification densities ranging from 1 adduct per100 bases (at 9.6 pg/ml 4'-AMDMIP) to 1 adduct per 1 1 bases(at 359 jg/ml) can easily be attained. This data indicates the rangethat can be achieved when 4'-AMDMIP is reacted with complexDNA and shows that the modification density can be controlledby the concentration of this photoreagent. At approximately 100pAg/ml of 4'-AMDMIP, a modification density of 1 adduct per15 bases was observed. With AMIP at a similar concentration,a modification density of 1 adduct per 33 bases was obtained.With 5-MIP, which has a solubility limit of 10 ytg/ml, the highestmodification density observed was 1 adduct per 88 bases.Although 5-MIP adducts are effective stops for Taq polymerase,this compound is inadequate as a sterilization reagent becauseits solubility limit prevents high modification densities.The important parameter to optimize sterilization is the average

number of adducts per amplicon strand. A robust post-PCRsterilization protocol would ensure that a major spill of a PCRreaction mixture would not lead to a carryover problem. Toensure that all the strands in a plateau PCR reaction mix (approx.6x 1011 amplicons/100 ul ) contain at least one adduct, Poissonstatistics require a minimum average of 28 effective adducts peramplicon strand. For the 1 1Smer, this requirement is satisfied

106 Nucleic Acids Research, Vol. 19, No. I

TabIle IL: ExpectednIlLliihcI-ftnion-sterilizedi alllll) iconl mlll)lecIles aIs aI I'LinctionIt P'(R PrlTidCt length.

CASEA (1 adduct per 5 bases)

Length of 'Average Effective Non-Sterilized MoleculesPCR Product Adducts/Strand per 6 x 1 011 Starting Molecules

1 001 50200250300

1 020304050

2.7 x 1071.2 x 103

1

.1 1

QASE B: (1 adduct per 15 bases)

250300350400450

13.316.62023.326.6

9.7x 1053.4 x 1041.2 x 103

4.41 .5

Assumes that to be effective, the adducts must be in the segment of the PCRproduct that is bounded by the primers. For calculation purposes, the primerlengths were taken to be 25 bases each.

VolurTe ofCarryovLriLl .i

1 oi10)

0 1

1,0-

o

1 C...'

1 C, -4

1 C;

10'

RANGE OF STERILIZATIONYWITHI ISOPSORALENS

Effective Adducts Per PCR Strandas as as 15

A) 115-mer ReamplificationI<.1 8 M specific PCR product

B) 500-mer Reamplitication

6xl 13 6x106 6x104 6X102

1 2 3 4 5 6 7 8

........

.....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:..

* ': :.

iop;es ofCarryover 30100 3x100 3x10430104h4v t +

2 3 4 5 6 7 a

Copies ofCarryover

Lane

Figure 4. Comparison of 4'-AMDMIP sterilization of a 115mer amplicon tosterilization of a 500mer amplicon. The reamplification of sterilized and non-sterilized amplicons are shown in the autoradiograph. The upper panel correspondsto reamplification of 1l5mer amplicon while the lower panel corresponds toreamplification of the 500mer amplicon. Lanes 1, 3, 5, and 7 are reamplificationsof the non-irradiated controls. Lanes 2, 4, 6, and 8 are reamplifications fromsterilized amplicons.

at an average modification density of 1 adduct per 2 bases.Although the modification density is controlled by the isopsoralenconcentration, it is not possible to achieve such high modificationdensities. At concentrations exceeding 200 jig/ml of

Figure 5. Summary of sterilization effectiveness as a function of modificationlevel of PCR strands and comparison to direct detection of carryover molecules.Beneath the columns of effective adducts per strand, the height of the gray areaindicates the number of carryover molecules for which it is statistically unlikelythat a single amplicon is without at least one effective isopsoralen adduct. Thesignal in the direct hybridization column was calculated as described in Table I.

4'-AMDMIP, PCR amplification of the 1l5mer, especially atlow copy number, begins to be inhibited (14). We havedetermined that the inhibition is sequence dependent and that theconcentration of isopsoralen that causes the inhibition varies witheach amplicon system and each isopsoralen derivative (G.D.Cimino, unpublished observations). While this inhibition can bealleviated by the addition of a co-solvent such as DMSO orglycerol(14), the hybridization efficiency of amplicons modifiedby 4'-AMDMIP also diminishes with increasing 4'-AMDMIPconcentration (G.D. Cimino, unpublished observations). When4'-AMDMIP is used in excess of 200 jig/ml, a significant dropin hybridization efficiency of the modified 1l5mer ampliconoccurs. If the detection protocol does not require the modifiedamplicon to hybridize to another probe molecule, this latter effectis not an issue and higher concentrations of isopsoralen (orpsoralen) can be used.For a fixed modification density there is an alternative method

for improving amplicon sterilization with photochemically activereagents. By choosing PCR primers judiciously, the length ofthe PCR products can be varied, and therefore, the averagenumber of adducts per strand can be varied. Table II illustratesthis effect for two different modification densities. In Case A,a modification density of 1 adduct per 5 bases is evaluated. Underthese conditions a 200 base long PCR product should haveapproximately 30 effective adducts per strand. At this level ofmodification, it is statistically unlikely that a single amplicon willhave escaped modification; all of the potential carryovermolecules should be sterilized. As pointed out earlier, we haveexperimentally determined that this level of modification densitycan be achieved with concentrations of 4'-AMDMIP that arecompatible with hybridization detection formats and withamplicons that have a very high AT content. Case B in Table

ASSL;vriyEc

c.

L. .3 Fl e

Nucleic Acids Research, Vol. 19, No. 1 107

II considers the more general situation with most amplicons wherethe modification density is taken, conservatively, to be 1 adductper 15 bases. Under these conditions, the same level ofsterilization will require an amplicon that is in excess of 450 basesin length.We have experimentally evaluated the length dependency for

4'-AMDMIP sterilization of PCR amplicons. Figure 4 comparesthe sterilization level of the 1 i5mer amplicon to the sterilizationlevel of a longer 500mer amplicon. The 1 i5mer reamplificationseries demonstrates that some, but not all, of the amplicons wererendered incapable of acting as carryover templates. By contrast,reamplification of the irradiated 500mer series did not result inany detectable PCR product for the highest copy numberexamined (6 x 108 copies). This experiment used a relativelylow, non-optimal concentration of 4'-AMDMIP to illustrate thelength dependence phenomena. However, as described in theaccompanying manuscript, the use of optimal concentrations of4'-AMDMIP provides highly effective sterilization of the 115-meramplicon.

SUMMARY

Several monoadducts of isopsoralen derivatives were shown tostop polymerization reactions with Taq DNA polymerase. Theuse of isopsoralens as photochemical post-PCR sterilizationreagents requires that the statistical nature of the modificationprocess be matched with the level of amplification and the lengthand sequence of the amplicon. We have shown that effectivesterilization is an operationally defined term. It implies thatcarryover cannot be detected under a precisely defined set ofamplification and detection conditions. For optimum results itis therefore necessary to design and evaluate a post-PCRsterilization protocol for each amplicon system.The design procedure should begin with an assessment of the

tolerable level of carryover so that the length and sequence ofan amplicon can be matched with a practical level of isopsoralenconcentration. Figure 5 correlates different volumes of PCRcarryover with the number of effective adducts necessary to bestatistically confident that the carryover event does not containa non-sterilized amplicon. 15 average adducts per PCR strandare sufficient to deal with carryover originating from 10-4 U1or less. If the carryover event results from larger volumes, moreeffective adducts per PCR strand are necessary. A practicalsterilization procedure should be effective at least to a level equalto the routine contamination. In our experience, the magnitudeof the carryover problem is much less than 107 molecules perevent (typically 10 to 104 molecules). An additionalconsideration is a second type of false positive PCR signal whichexists when carryover exceeds 107 amplicons. These ampliconsare detected directly by hybridization to a 32p probe. Therefore,while it is desirable to design a sterilization protocol capable ofsterilizing all amplicons in a PCR reaction tube, it is not necessaryto achieve levels of sterilization that exceed direct detection ofthe carryover molecules themselves.The above considerations will determine a range of isopsoralen

concentrations that need to be appraised for their sterilizationability and for their effect on detection sensitivity. PCRamplification efficiency and, where appropriate, hybridizationefficiency contribute to detection sensitivity. Amplificationefficiency in the presence of isopsoralen should be evaluatedunder conditions that are far away from PCR plateau so thatinhibitory effects can be seen. Using tolerable concentrations ofisopsoralen, the hybridization efficiency of the modified

amplicons should then be determined. Finally, the number ofPCR cycles should be adjusted to bring the detection level backto the level that exists in the absence of the sterilization process.By following these steps, an effective and practical post-PCRsterilization protocol will emerge that eliminates the carryoverproblem associated with the PCR. The accompanying paperillustrates the design and refinement process of a post PCR-amplification sterilization protocol for the detection of low copynumbers of a retroviral sequence in clinical samples (14).

ACKNOWLEDGEMENTS

We thank Dr. John Sninsky and Shirley Kwok for providingDNA from an HIV positive patient and for many helpfuldiscussions, and Dr. Corey Levenson, Lori Goda and DraganSpasic for providing oligonucleotides. This work was supportedin part by a contract from Cetus Corporation.

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