RAPID COMMUNICATION |
Correspondence to: G.J. Nuovo, MGN Medical Res. Laboratory, 8 Huckleberry Lane, Suite 5, Setauket, NY 11733.
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Summary |
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We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used. (J Histochem Cytochem 47:273279, 1999)
Key Words: in situ PCR, PCR, hepatitis C, HPV, HIV-1
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Introduction |
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The in situ amplification of DNA or RNA is useful in situations characterized by low copy number of the target. Point mutations, latent viral infection, and the low copy number that characterizes many viral and eukaryote mRNAs are examples in which the detection of a DNA or RNA target is augmented by in situ amplification of the nucleic acid sequence of interest (
Conventional PCR-based assays include analysis of the amplified DNA using gel electrophoresis or hybridization. Recently, several methods have become available that permit one to monitor the DNA amplification directly in the reaction mixture without separation of the amplified DNA from the unreacted primers (
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Materials and Methods |
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For HPV analysis by in situ PCR, CaSki (500 copies HPV-16 per cell) and SiHa (one copy of HPV-16 per cell) were grown to confluence and fixed for 13 days in 10% buffered formalin as previously described (
The samples were digested in 2 mg/ml pepsin for 3090 min. The protocol used for detection of HPV-16 DNA by in situ amplification has been described (
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Our protocol for the detection of hepatitis C RNA using RT in situ PCR has been described (
Solution-phase PCR was performed at four different settings using (a) two conventional linear primers, (b) one conventional linear primer and energy transfer-labeled hairpin primer with the 3' sequence complementary to the target, (c) one conventional linear primer, one tailed primer, and a UniPrimer, or (d) two tailed primers and a UniPrimer. Amplification was performed in 20 µl as described previously (
A Shimadzu RF-5000 spectrofluorophotometer and Victor fluorescent platereader were used to measure the fluorescence. For the spectrofluorophotometer, 2.55 µl of the reaction mixture was diluted to 600 µl with 20 mM Tris-HCl, pH 8.5, 50 mM NaCl, 2 mM MgCl2, and emission at 516 nm with 490-nm excitation was detected in the 10 x 3 cuvette. For the platereader, the detection was performed directly in closed 0.2-ml PCR tubes.
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Results |
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In Situ PCR with the UniPrimer System
To validate the specificity of in situ PCR using the HPV-16-specific UniPrimer system, the Caski (500 copies HPV-16/cell) and HeLa (HPV-18-positive) cells were examined. HPV-16 DNA was readily detected in the CaSki cells by in situ PCR using direct incorporation of the UniPrimer with the HPV-specific primers. No signal was evident in the HeLa cells. Equivalent results were obtained using one- or two-tailed target-specific primers (Table 1) or with primers that contained one biotin or digoxigenin molecule per oligomer, reflecting the high target copy number in these cells (data not shown).
We next studied the conditions that were needed to detect low-copy targets using in situ PCR and labeled primers. SiHa (1 copy HPV-16/cell) cells were used. No signal was evident when the primers labeled with either one biotin or one digoxigenin molecule were used. A signal was seen in 10% of the SiHa cells when the primers that contained three biotin moieties per oligomer were used. This compares to a detection rate of 100% if PCR in situ hybridization is used (Figure 2). A signal was evident in 10% of the SiHa cells when the UniPrimer system with one tailed primer was used (Figure 2). When both HPV-specific primers were tailed, the percentage of SiHa cells with detectable signal increased to 100% (Figure 2). The latter signal was lost when either the Taq polymerase or magnesium was omitted from the amplifying solution. Therefore, in situ PCR using two-tailed primers with the UniPrimer was as sensitive as PCR in situ hybridization using a labeled probe, being able to reliably detect one target copy per cell.
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Next, four cervical SILs, each of which contained HPV-16 from low copy, detectable only after in situ hybridization, to high copy numbers were examined with the two tailed primers (i.e., each target-specific primer labeled with sequence whose complement could hybridize with the UniPrimer) and compared to the data generated on serial sections from PCR in situ hybridization (with a labeled probe) and in situ PCR using the primers with three biotin molecules per oligomer. A strong signal was evident in each of the four cervical tissues that localized to the dysplastic cells only, which confirms its specificity, and was equivalent when PCR in situ hybridization with a labeled probe was compared to in situ PCR with the two-tailed UniPrimer system. In each case, the number of positive cells was greater compared to standard in situ hybridization. However, the number of positive cells decreased to below that noted for standard in situ hybridization when in situ PCR was done using the HPV-16-specific primers that contained three biotin molecules per primer (data not shown).
Next, we studied the utility of the UniPrimer system for RT in situ PCR. The comparison was made between RT in situ PCR after DNase digestion and direct incorporation of the labeled nucleotide vs RT in situ PCR with no DNase digestion and in which the equivalent primers were modified for use with the UniPrimer system. Five liver biopsies that contained 550 or more infected hepatocytes were studied, as well as two liver tissues negative for hepatitis C (and positive for hepatitis E and G, respectively). In each of the five cases, an equivalent number of hepatocytes were positive using either RT in situ PCR with DNase digestion and digoxigenin dUTP or RT in situ PCR without DNase digestion and the UniPrimer system (Figure 2). With respect to the UniPrimer system, no signal was evident if the primers were omitted or in the two negative controls (Figure 2). A strong signal was evident with the digoxigenin-labeled nucleotide when the primers and DNase step were omitted, owing to DNA repair, as described previously (
Solution-phase PCR with the UniPrimer
First, the sensitivity of solution-phase PCR using standard primers was compared to that using the energy transfer-labeled primers with the HIV-1 gag region as the model system. Three different primer sets were tested: (a) two conventional linear primers, (b) one conventional linear primer and energy transfer-labeled hairpin primer with the 3' sequence complementary to the target, and (c) one conventional linear primer, one tailed primer, and the UniPrimer. To compensate for the first cycles in which UniPrimer was not incorporated, three more cycles were performed in PCR with the UniPrimer compared to the other two systems. The results in Figure 3 for HIV-1 demonstrate that the UniPrimer-based system yielded the PCR product of the expected size and that the yield is comparable with that for the regular primers and the energy transfer-labeled target-specific hairpin primers.
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Next, we studied whether the use of two-tailed primers had any effect on the specificity of solution-phase PCR. In these analyses, both specific primers have the same tail that dictates the synthesis of the corresponding sequence which is complementary to the initial region of the UniPrimer, which permits the latter to incorporate into both ends of the PCR product. Therefore, both tailed target-specific primers are used in concentrations one tenth of that of the UniPrimer, because they are necessary only during the first step of the reaction. It is possible that this may affect primer oligomerization, a common PCR pathway that can interfere with target-specific amplification (
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Discussion |
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The main finding of this study was that one could detect one copy per cell with in situ PCR using two tailed primers and the corresponding UniPrimer sequence. This improved sensitivity with in situ PCR may relate to decreased primer oligomerization, as demonstrated by solution-phase PCR with the corresponding UniPrimer system. The use of labeled primers enables one to do target-specific incorporation for detection of DNA targets with hot-start in situ PCR without the need for a hybridization step. This is especially important with hybridization using an oligoprobe, because of the narrow window between signal and background and the concomitant potential for weakened signal and/or background (
The foundation of the AmpliFluor UniPrimer system is the hairpin structure located on its 5' end. Two moieties are attached to the stem sequence of the hairpin: a fluorophore (fluorescein) on the 5' end, and a quencher (DABSYL) on the opposite side of the hairpin stem. DABSYL is a nonfluorescent chromophore whose absorption spectrum overlaps the emission spectrum of fluorescein. When stimulated by light of peak wavelength 488 nm, fluorescein emits fluorescence of peak wavelength 516 nm. However, when DABSYL is located sufficiently close to the donor fluorophore (less than 100 Angstroms), the energy from the excited fluorophore can be transferred to DABSYL and dissipated as heat. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching of the donor fluorophore. In the early steps of amplification, the extension of the labeled target-specific primers 1 and 2 will yield an amplicon for which the 3' end of each strand is complementary to a corresponding region of the UniPrimer. During the amplification reaction, the hairpin structure of the UniPrimer is unfolded and copied and the fluorescein and DABSYL are no longer close enough to permit quenching. Instead, a fluorescent signal is emitted. To minimize the competition between the tailed primers and the UniPrimer, the former are used in a much lower concentration. As a result, the majority of the PCR product has the incorporated hairpin primer and generates the fluorescent signal.
An amplification detection system based on incorporation of energy transfer-labeled primers is an efficient way to eliminate carryover contamination and to simplify the high throughput detection procedure (
For any PCR-based system, the absence of side reactions that may generate a background signal is crucial. One of the common PCR artifacts is primer oligomerization (
The UniPrimer-based methodology described here circumvents a major problem with the in situ PCR technique using paraffin-embedded tissues. Specifically, the heating to 65C for 4 hr that is obligatory in paraffin-embedded tissues induces DNA nicks (
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Acknowledgments |
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Supported by NIH SBIR grant 1R43GM56045.
We would like to thank Yuri Khripin for valuable discussions and Laura Weihrauch for technical assistance.
Received for publication July 13, 1998; accepted November 3, 1998.
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