Optimal Activation of Isopsoralen To Prevent Amplicon Carryover

Abstract

We compared the efficiencies of activation of the photochemical isopsoralen compound 10 and its resulting amplicon neutralizations under conditions with a UV transilluminator box at room temperature (RT) and a HRI-300 UV photothermal reaction chamber at RT and at 5°C. Our data suggest that use of the HRI-300 reaction chamber at 5°C results in a statistically significantly higher degree of amplicon neutralization.

PCR technology has become a powerful and extremely sensitive tool in a broad range of research and diagnostic applications. Yet ironically, it is this extreme sensitivity that makes PCR very prone to false-positive results. Repeated amplification of specific target DNA sequences results in the accumulation of intact and degraded PCR products (amplicons) and primer artifacts that can contaminate subsequent amplification reactions (1, 9).

A major source of this carryover contamination is the aerosols created while manipulating PCR amplicons (12). Even when following strict laboratory technique (6–8, 13), including physical separation of pre- and postamplification procedures, it has been recommended that the reaction vessel never be opened to the environment without prior application of a sterilization procedure (5).

One such method of sterilization utilizes the photochemical isopsoralen compound 10 (IP-10). IP-10 is added to the reaction mixture prior to amplification. Following PCR but before the reaction tube is opened, the vessel is exposed to UV light (300 to 400 nm), which activates the IP-10 to form adductors between the pyrimidines on the amplicons (10). These adductors stop Taq polymerase from processing along the amplicons and thus prevent subsequent reamplification of any of these contaminating amplicons.

Several articles describing the effects of concentration of IP-10 and the length and sequence of the amplicons on sterilization efficiency have been published (2, 3, 5, 11). However, to date, no publication has examined the effects of various sources and conditions of UV light on the efficiency of IP-10 activation.

(These data were presented previously [4].)

By the PCR protocol described below with primers targeting the cytomegalovirus (CMV) glycoprotein gene, a 209-bp sequence was amplified. A solution of stock amplicons was produced by pooling six separate PCR mixtures, each inoculated prior to amplification with approximately 2,000 virions of the CMV reference strain AD169. The stock solution was measured on a Turner Digital Fluorometer (Barnstead/Thermolyne, Dubuque, Iowa) and the number of amplicon copies per microliter was calculated. The stock solution was then divided into four separate aliquots. One aliquot (referred to as No UV) was not exposed to any UV light source. One aliquot (referred to as UV Box) was exposed to UV light by placing the tube on a transilluminator box (Fotodyne, Inc., Hartland, Wis.) for 15 min. The other two aliquots were exposed to UV light for 15 min by placing the tubes in an HRI-300 photothermal reaction chamber (Simms Instruments, Palo Alto, Calif.), one at room temperature (RT) (referred to as HRI-RT) and one at 5°C (referred to as HRI-5°C). The measured temperature for the RT condition was 25 ± 2°C; the measured temperature for the 5°C condition was 5 ± 2°C. A dilution series with TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) was then performed on each aliquot, and two 5-μl samples from each dilution were subjected to reamplification. The dilution series and reamplification were performed in triplicate on each of the four aliquots.

Both initial and reamplification PCRs were performed in a mixture of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM deoxynucleoside triphosphates, 2.5 U of Taq polymerase (Roche Molecular Systems, Inc., Branchburg, N.J.), 0.5 μM each primer (Research Genetics, Inc.), 25 μg of IP-10 (HRI Research, Inc., Concord, Calif.)/ml, 2.5 mg of bovine serum albumin (GibcoBRL)/ml, and 10% glycerol (Mallinckrodt Specialty Chemicals Co., Paris, Ky.) in a total volume of 50 μl. The forward (CMV GB1-BIO) and reverse (CMV GB2) primer sequences were 5′-biotin-CGC-TCG-CTG-CTC-TGC-GTC-CAG-ACG-GG-3′ and 5′-CCG-CCG-ACG-GGA-CCA-CCG-TGA-CG-3′, respectively. The IP-10 concentration used was based on manufacturer recommendations for the length and GC content of the expected PCR amplification product. The reaction tubes were incubated in a thermal cycler (DNA Engine PTC-200; MJ Research, Inc., Watertown, Mass.). After an initial denaturation period of 5 min at 94°C, a two-step program—consisting of 94°C for 30 s and 72°C for 2 min for a total of 38 cycles and concluding with a final extension period of 5 min at 72°C—was used.

From each of the reamplification reaction mixtures, a 5-μl aliquot was added to a streptavidin-coated microdilution well containing 100 μl of assay buffer (Wallac Oy) supplemented with 1 M NaCl. The plate was shaken at a low speed on a DELFIA Plateshake device (model 1296-0022; Wallac Oy) for 30 min at RT. Since the CMV forward primer was modified with a biotin label, the resulting biotinylated amplicons become bound to the streptavidin. The buffer was then aspirated by using a Columbus (Wallac Oy) platewasher (SLT-Labinstruments GmbH, Grödig, Austria), and 100 μl of 50 mM NaOH was added to each well. The plate was then incubated at RT for 10 min to denature the captured amplicon, and the wells were washed twice with 1× wash buffer (Wallac Oy) to remove the unbound strand. The Eu³⁺-labelled target probe, Eu³⁺-GCA-CCA-AAG-ACA-CGT-CGT-TAC-AGC-C (Wallac Oy), was diluted in assay buffer supplement with 1 M NaCl and 1× blocking reagent (Boehringer Mannheim, Indianapolis, Ind.) to obtain a final concentration of 0.02 ng/μl. A 100-μl aliquot of this dilution was added to each well, and the plate was sealed with a plastic cover and incubated for 2 h at 55°C. After hybridization, the wells were washed five times with 1× wash buffer. A 200-μl aliquot of enhancement solution (Wallac Oy) was added to each well, and the plate was shaken at low speed for 25 min at RT. Fluorescence was then measured on the DELFIA 1234 time-resolved fluorometer (Wallac Oy). Six reamplification PCR assays were performed on each of the dilutions of stock amplicons pretreated with the various conditions. The DELFIA hybridization assay was performed on each replicate, and the mean time-resolved fluorescence value and resulting P values were calculated by using the StatView 4.1 program.

Based on previous studies (data not published), applying a cutoff value of 15,000 to the DELFIA hybridization assay yields a sensitivity comparable to that of the Southern blot hybridization procedure utilizing a chemiluminescence-labelled probe.

Table 1 compares the reamplification activity as determined by mean time-resolved fluorescence values of the amplicons after exposure to the various test conditions. Prior to exposure to UV light, IP-10 is not active and all amplicons are available for reamplification. Therefore, comparison of the mean values for the No UV dilution series to those from the UV Box dilution series produces P values that are all highly significant (<0.0001). To evaluate the efficiency of IP-10 activation from two different UV light sources at RT, the mean fluorescence values obtained from the UV Box and HRI-RT dilution series were compared. The mean values obtained from the HRI-RT dilution series were all much lower than those obtained from the UV Box dilution series, although the differences in the lowest two dilutions were not statistically significant (P > 0.05). To determine if temperature had an effect on the efficiency of IP-10 inactivation of amplicons, the HRI-RT and HRI-5°C dilution series mean values were compared. All dilutions from the HRI-5°C series produced statistically significantly lower mean fluorescence values. Although it is commonly available in most laboratories, use of the UV transilluminator box for activation of IP-10 must be done with caution, as our results show that at RT it does not inactivate even as few as 2.6 × 10⁴ amplicons effectively. The increased IP-10 activation obtained using the HRI-300 chamber is most likely due to the dramatic difference in the amount of UV energy delivered to the tubes. Due to suspension of the tubes in the holding rack and the reflective properties of the internal chamber of the HRI-300, the manufacturer has determined that the net UV energy delivered to each transparent sample is approximately 27 mW/cm². Conversely, the measured output from the UV box was only 1.2 mW/cm², as measured by a Blak-Ray UV intensity meter (model J-221; UVP, Upland, Calif.). The HRI-300 chamber costs approximately $2,000.00 (mid-1998 pricing) and must be either attached to a cooling water bath (additional cost) or placed in a refrigerator to achieve a cooled environment for optimal IP-10 activation.

TABLE 1.

Comparative reamplification activity as determined by mean time-resolved fluorescence values^a

Amplicon copies per new PCR	Mean time-resolved fluorescence values (P value) for:
	No UV/UV Box	UV Box/HRI-RT	HRI-RT/HRI-5°C
1  × 1010	5,826,220/1,120,853 (<0.0001)	1,120,853/701,728 (0.0008)	701,728/398,821 (0.0005)
2  × 109	5,995,315/817,178 (<0.0001)	817,178/383,279 (0.0005)	383,279/152,328 (0.0002)
4  × 108	6,053,433/634,798 (<0.0001)	634,798/235,957 (0.0006)	235,957/121,685 (<0.0001)
1.6  × 107	5,942,940/416,950 (<0.0001)	416,950/141,336 (0.0037)	141,336/50,147 (0.0003)
6.5  × 105	5,412,027/269,447 (<0.0001)	269,447/85,642 (0.0067)	85,642/34,958 (0.0235)
1.3  × 105	5,815,833/111,106 (<0.0001)	111,106/54,681 (0.1539b)	54,681/13,052 (0.0003)
2.6  × 104	4,987,103/43,798 (<0.0001)	43,798/24,345 (0.2901b)	24,345/1,569 (0.0042)

^aSix replicates were performed for each condition. Results are presented as pairwise comparisons and P values (by paired t test; calculated with StatView) of aliquots of amplicons exposed to various test conditions. 

^bNot significant (P > 0.05). 

Our results suggest that an HRI-300 photothermal reaction chamber provides a more efficient method to activate IP-10 than does a UV transilluminator box. In addition, activation of IP-10 at 5°C results in better neutralization of amplicons than activation at RT when the same UV light delivery system is used. IP-10 offers a simple and effective method to neutralize potentially contaminating amplicons. Depending on the mode and temperature of IP-10 activation, IP-10 can effectively prevent carryover contamination of as many as 1.3 × 10⁵ amplicons.