Created: 28th February 1999, last updated: 7th April 1999, © 1999 ABRF
Deborah S. Grove
Nucleic Acid Facility, Life Science Consortium, The Pennsylvania State University, University Park, PA 16802
Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems Division 7700 Sequence Detector provides an accurate method for determination of levels of specific DNA and RNA sequences in tissue samples. It is based on detection of a fluorescent signal produced proportionally during amplification of a PCR product. Turn-around time for data acquisition and analysis by real-time PCR with the 7700 model is short, and results are more reliable than by traditional PCR methods. This technology can be successfully offered as a service in a core faculty setting. (J Biomol Tech 1999;10:11-16)
Key words: quantitative real-time polymerase chain reaction, Perkin Elmer/Applied Biosystems 7700 Sequence Detector, nucleic acid sequence quantification.
Address correspondence and reprint requests to Deborah S. Grove, Nucleic Acid Facility, Life Science Consortium, Pennsylvania State University, University Park, PA 16802.
Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems (PE/ABD) (Foster City, CA, USA) Prism 7700 Sequence Detector System is a relatively new technology that provides a broad dynamic range (at least five orders of magnitude) for detecting specific gene sequences with excellent sensitivity and precision. DNA and RNA can be quantified using this detection system without laborious post-PCR processing.1,2 This technology can be performed in a core facility environment because of the costs associated with instrument purchase, setup, and maintenance.
Quantitative real-time PCR using the PE/ABD 7700 is based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product. The chemistry is the key to the detection system (Fig. 1). A probe (ie, TaqMan) is designed to anneal to the target sequence between the traditional forward and reverse primers. The probe is labeled at the 5' end with a reporter fluorochrome (usually 6-carboxyfluorescein [6-FAM]) and a quencher fluorochrome (6-carboxy-tetramethyl-rhodamine [TAMRA]) added at any T position or at the 3' end.3 The probe is designed to have a higher Tm than the primers, and during the extension phase, the probe must be 100% hybridized for success of the assay. As long as both fluorochromes are on the probe, the quencher molecule stops all fluorescence by the reporter. However, as Taq polymerase extends the primer, the intrinsic 5' to 3' nuclease activity of Taq degrades the probe, releasing the reporter fluorochrome.4 The amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle.
FIGURE 1. Fluorogenic 5' nuclease chemistry. (1) Forward and reverse primers are extended with Taq polymerase as in a traditional PCR reaction. A probe with two fluorescent dyes attached anneals to the gene sequence between the two primers. (2) As the polymerase extends the primer, the probe is displaced. (3) An inherent nuclease activity in the polymerase cleaves the reporter dye from the probe. (4) After release of the reporter dye from the quencher, a fluorescent signal is generated.
In brief, the 7700 detection system consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system. An optical fiber inserted through a lens is positioned over each well, and laser light is directed through the fiber to excite the fluorochrome in the PCR solution. Emissions are sent through the fiber to the CCD camera, where they are analyzed by the software's algorithms. Collected data are subsequently sent to the computer. Emissions are measured every 7 seconds.
The sensitivity of detection allows acquisition of data when PCR amplification is still in the exponential phase. This is determined by identifying the cycle number at which the reporter dye emission intensities rises above background noise; this cycle number is called the threshold cycle (Ct) (Fig. 2). The Ct is determined at the most exponential phase of the reaction and is more reliable than end-point measurements of accumulated PCR products used by traditional PCR methods. The Ct is inversely proportional to the copy number of the target template; the higher the template concentration, the lower the threshold cycle measured.
FIGURE 2. One of the views available after completion of the run is an amplification window. This window shows the amount of fluorescence obtained in each amplification cycle for each reaction. The threshold cycle (Ct) is shown by the darker horizontal line.
There are many advantages to quantifying gene sequences using this technology, foremost being sensitivity and precision. This precision exists because quantitation of the gene sequence is determined by the Ct, which is calculated during the exponential phase of the reaction. High specificity is conferred by the requirement of three oligos to anneal to the DNA before any data are collected.
Turn-around time for data acquisition and analysis by real-time PCR with the 7700 is short. Setup and thermal cycling require less than 3 to 4 hours and data analysis less than 10 additional minutes. Traditional PCR quantification may require several days.
Competitive PCR is another technique often used to quantify DNA or RNA. Optimization of competitive PCR is laborious and time consuming. Several dilutions of target sequences must be tested to achieve a suitable ratio of target to competitor, and efficiencies of target and competitor must be similar. This assay is linear only over a very short range compared with quantitation with the 7700. The number of samples that can be processed is also a limiting factor.
An advantage of providing the quantitative real-time PCR technology in a core facility setting is reduction of labor time and costs. One half-time employee can easily process 96 samples per day, including setup and data analysis. The use of robotics often found in core facilities allows preparation of samples with speed and accuracy.
The applications for quantitative real-time PCR are innumerable. Detection of genomic or viral DNA in tissues can be a valuable diagnostic tool. Gene expression can be measured after extraction of total RNA and preparation of cDNA by a reverse transcription (RT) step. Setup and analysis are simple and can more easily be extended to the clinical environment than traditional PCR techniques.
One problem with developing applications is that most research conducted with this technology is done by pharmaceutical companies. Because their work remains proprietary, successful TaqMan primers and probes, as well as optimization conditions, are often not available. As a result, de novo design and optimization still require an investment of time and money.
Examples of gene sequences that have been optimized for detection are shown in Table 1. My colleagues and I also have optimized detection systems for prolactin, prolactin receptor, perforin, five isozymes of plant 1-aminocyclopropane-1-carboxylic acid synthase (ACS), type X collagen, cartilage matrix protein, occludin, JC virus, and CD19 (unpublished results).
Reported Real-Time Polymerase Chain Reaction Gene Sequences
|DNA-dependent protein kinase||5|
|beta-Actin, interleukin (IL)-2, IL-4, IL-10, interferon-gamma||6,7|
|Tumor necrosis factor-alpha||6|
|JH and VH genes of B cells||9|
|CDR3 sequences for multiple myeloma clones||9|
|SRY fetal protein||14|
|Herpes simplex thymidine kinase||15|
|AML1/ETO leukemia protein||16|
|Philadelphia chromosome-positive leukemia||17|
|Plasma simian immunodeficiency virus||18|
|E. coli toxin genes||20|
|Mycobacterium tuberculosis DNA||25|
|Swine fever virus||27|
Although I have focused on gene detection in this article, another application is an allelic discrimination assay that can detect single-base nucleotide mutations and polymorphisms. These assays require two separate probes that differ only by one base mismatch. One probe labeled with 6-FAM represents one allele, and the other probe labeled with the fluorochrome VIC represents the other allele. A mismatch leads to a less efficient amplification. Fluorescence spectra are collected after the run, and using multicomponent analysis, the software extracts the contribution of each component dye to the observed spectrum. Homozygotes for FAM show an increase in the FAM signal but no increase in the VIC signal, and homozygotes for the VIC probe show an increase in that signal. Heterozygotes show intermediate increases of FAM and VIC signals. All three groups are clearly distinguishable,28 and the sensitivity is similar to that for the quantitative PCR application.
Optimization of the PCR reaction is required for each primer and probe set. The optimal Mg2+ concentration is usually between 4 and 6 mM but sometimes can be as low as 2 mM. Optimal primer concentrations are usually between 100 and 800 nM. Optimization requires varying the concentration of one primer relative to the other, because the optimal concentration may not be the same for both. The optimal probe concentration may be as low as 50 nM or as high as 200 nM. The optimal Mg2+ concentration and reverse primer concentration must also be validated for the RT step.
The detection system is so sensitive that fewer than 10 copies of DNA can be detected. Aerosol contamination of primers and probes is a potential problem if samples are prepared in the laboratory where DNA is being extracted. Probes synthesized by outside companies are delivered directly to us. We synthesize and cartridge-purify our own primers, and we prepare all PCR or RT-PCR reactions in a laminar flow hood. After thermal cycling, the sample plate is kept closed until analysis to minimize the chance of releasing PCR products.
For determination of gene expression, total or polyA RNA is isolated. A specific cDNA can be produced by using the same reverse primer used in the PCR reaction or by using random hexamer primers to produce a range of cDNA products. RNA can easily be prepared using kits such as RNAEasy from Qiagen (Valencia, CA, USA) and Triazol from Life Technologies (Gaithersburg, MD, USA).
Multiplexing quantitative PCR reactions by using more than one fluorescent dye per tube was not an option when the 7700 was first released. Because we could not use internal tube controls, we used the ribosomal phosphoprotein (RPPO) as an external control and found the data obtained to be consistent. After multiplexing became available, internal tube controls could be established. Kits are available from PE/ABD for 18S ribosomal RNA or for glyceraldehyde-3-phosphate dehydrogenase as a control. We have found that 18S appears to work across many species, including plants. The probe used for the internal control has been labeled with the PE/ABD fluorochrome, JOE. A fluorochrome with a higher intensity, VIC, is also available. These two fluorochromes are preferred for use with FAM, the reporter used on the probe.
If copy number is required, standard curves of plasmid DNA can be constructed and assayed each time with samples containing the target gene sequence. If the starting molecule is RNA, cRNA can be prepared and used as a standard. Kits are available to prepare RNA from plasmids containing the gene sequence. T7, T3, or SP6 primers typically are used to prepare the cRNA. The cRNA produced must be validated in the RT and PCR reactions to determine if it is transcribed and amplified at the same efficiency as the sample RNA present in a mixture of extracted RNAs.
Other important controls are no-amplification controls (NACs) and no-template controls (NTCs). NACs test for contamination of RNA by genomic DNA. If the primers and probe set have been designed so that at least one spans an intron, this control is unnecessary. However, intron sites may not be known or may not be present. Nearly all RNA samples that we have received have had NACs with Cts at least three cycles higher and usually much more than three cycles. This has been acceptable for quantification. NTCs test for the contamination of assay reagents.
Several types of reaction mixes are available from PE/ABD. The TaqMan PCR Core Reagent kit has been satisfactory for our use and allows flexibility in preparing the different master mixes that are needed for the wide variety of primer and probe sets used. We mix the kit components to create a customized master mix for two-step RT-PCR that is easy to use and allows rapid assay development. A similar kit, TaqMan Universal PCR Master Mix, contains the core reagents in an easy to use 2X solution. The TaqMan Gold RT-PCR kit allows one-step or two-step RT-PCR. The one-step option allows an investigator to set up the RT and PCR steps without opening the tube, whereas the two-step option separates the RT step from the PCR. The two-step protocol is used for assays for which multiple transcripts are being prepared from random hexamers or oligo-dT. The tube is opened after the RT step, and aliquots are removed and run with specific TaqMan primers and probes. The TaqMan EX RT-PCR kit specializes in applications that require a higher-temperature RT step to destabilize secondary structure.
Master mixes can also be assembled by purchasing the various components, such as NTPs, buffer, Mg2+, and Taq polymerase, from many other companies offering molecular biology reagents. It is important to validate that only one specific PCR product is produced with the Taq polymerase selected.
Real-time monitoring of the release of fluorescence several times during each cycle allows collection of abundant data. After 40 cycles, data are processed by the software within seconds. Data can be viewed in an "amplification window" in the analysis program (see Fig. 2). This allows the operator to check the fluorescence from each reaction at each cycle. The linearity of the fluorescence response for each sample at each cycle and the baseline can be checked for each tube. An occasional problem tube can easily be identified and the data point discarded, or the amplification curve may indicate that a different baseline should be chosen for the experiment to generate more accurate Cts.
Primers and probes must be carefully designed because of the costs associated with producing probes with different dyes at 5' and 3' ends. We use PE/ABD Primer Express software, which is specifically designed to select the primers and probes. Considering the high numbers and varied backgrounds of potential users, our facility personnel designs primers and probes to ensure investigators' investment in expensive probes can lead to successful results.
The required parameters for well-designed primers and probe have been well documented and are built into the program.3 These parameters include a Tm for the probe that is 10°C higher than the primers, primer Tms between 58°C and 60°C, amplicon size between 50 and 150 bases, absence of 5' Gs, and primer length. If primers or probes are not found, these parameters can be modified with satisfactory results. Early versions of Primer Express designed primers and probe sets with longer amplicons, and some of these work very well.
The best design for primers and probes to use for the quantitation of mRNA expression requires positioning of a primer or the probe over an intron. The Primer Express software has a tool called Junction Annotation that allows a user to mark exon junctions. When the primers are calculated, at least one crosses an exon junction. Tools are also available to exclude certain regions or to choose certain regions for a probe or primer to be designed.
Primer and probe design is different for the allelic discrimination assays. The probes designed for each allele should be centered over the mismatched base, and the probes only have to differ by that base. Another major difference is that the probe for these assays has a lower Tm requirement than the TaqMan PCR assays.
Cost has been the biggest factor in determining whether investigators use this technology. We charge $4.50 per reaction starting with DNA and $5.40 per reaction starting with RNA. Kits can be purchased from PE/ABD to use for the entire procedure for the PCR reaction or RT-PCR. This is the least labor-intensive option and ensures quality control. However, master mixes for the reaction can be assembled by purchasing less expensive components such as NTPs, buffer, Mg2+, and Taq from other companies. We add RNAse inhibitor and reverse transcriptase to the Universal Master Mix to measure RNA, and this addition accounts for the higher price for reactions starting with RNA.
Labor costs vary among institutions. Our labor cost is about 10% of the final cost per reaction. Optical caps from PE/ABD, tubes, plates, and filtered tips comprise another 15% to 30% of the cost. The service contract cost (up to $8500 per year) can be as much as 10% to 20% of the cost per reaction, depending on number of samples submitted throughout the year. Cost-effective projects include those that involve medical or agricultural diagnostics and gene expression studies in clinical trials.
We typically advise investigators to order their TaqMan probes from outside vendors. Although we can easily synthesize the 5' 6-FAM derivatives on a standard DNA synthesizer, the 3' TAMRA labeling is more complex. The additional steps in the synthesis and post-synthesis purification can take 7 to 14 days to complete.
A solid support (TAMRA CPG) became available recently, allowing preparation of 3' TAMRA-labeled oligonucleotides in high yield. This may allow more rapid and purified synthesis of dual-labeled probes.
Two similar quantitative PCR instruments are available from PE/ABD that also measure the presence of DNA. The 5700 detects all light from 500 to 700 nm with no spectral separation, eliminating multiplexing and allelic discrimination assays. The 5700 and 7700 models have a linear dynamic range of 5 logs. The 7700 functional specification is twofold resolution using the beta-actin kit (5000 versus 10,000 copies), whereas the 5700 specification is fivefold resolution (2000 versus 10,000 copies). Both machines can use TaqMan or SYBR Green.
SYBR Green can be used as a "probeless" alternative to the TaqMan system. Because it binds to all double-stranded DNA, it is imperative to ensure that the PCR product being quantified is a clean, single product, because any primer-dimers or background smears are detected. It can be used as a prescreening assay before ordering a probe. If the results are satisfactory, the probe can be ordered and TaqMan reactions run with higher specificity.
The LightCycler from Boerhinger (Indianapolis, IN, USA) is another alternative. In a price range between the 7700 and 5700 models, it can do SYBR Green or TaqMan chemistry and perform analysis with hybridization probes based on fluorescent resonance energy transfer.
The LS-50B from PE/ABD is an end-point fluorescence detector used for allelic discrimination assays or "plus/minus" assays. The reactions are run in a thermal cycler and read in the LS-50B.
Quantitative real-time PCR using the PE/ABD 7700 Sequence Detector provides a rapid and accurate method for determining levels of specific DNA and RNA sequences in various tissue samples. This technology can be successfully offered as a service in a core faculty setting.
1. Gibson UEM, Heid CA, Williams PM. A novel method for real-time quantitative RT-PCR. Genome Res 1996;6:995-1001.
2. Heid CA, Stevens J, Livak KJ, Williams PM. Real-time quantitative PCR. Genome Res 1996; 6:986-994.
3. Livak KJ, Flood SJA, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995;4:357-362.
4. Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991;88:7276-7280.
5. Araki R, Funimori A, Hamatani K, et al. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency. Proc Natl Acad Sci USA 1997;94:2438-2443.
6. Kruse N, Pette M, Toyka K, Rieckmann P. Quantification of cytokine mRNA expression by RT PCR in sample of previously frozen blood. J Immunol Methods 1997; 210:195-203.
7. Casteels KM, Mathieu C, Waer M, et al. Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short course of cyclosporin A. Endocrinology 1998;139:95-102.
8. Luoh SM, DiMarco F, Levin N, et al. Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesin. J Mol Endocrinol 1997;18:77-85.
9. Gerard CJ, Olsson K, Ramanathan R, Reading C, Hanania EG. Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards. Cancer Res 1998;58:3957-3964.
10. Shimokawa T, Kato M, Ezaki O, Hashimoto S. Transcriptional regulation of muscle-specific genes during myoblast differentiation. Biochem Biophys Res Commun 1998;246:287-292.
11. Gerard CJ, Arboleda MJ, Solar G, Mule JJ, Kerr WG. A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses. Hum Gene Ther 1996;7:343-354.
12. Gelmini S, Orlando C, Sestini R, et al. Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erB-2 oncogene amplification. Clin Chem 1997;43:752-758.
13. deKok JB, Hendriks JCM, van Solinge WW, Willems HL, Mensink EJ, Swinkels DW. Use of real-time quantitative PCR to compare DNA isolation methods. Clin Chem 1998;44:2201-2204.
14. Lo YMD, Tein MSC, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-775.
15. Lockey C, Otto E, Long Z. Real-time fluorescence detection of a single DNA molecule. Biotechniques 1998;24:744-746.
16. Marcucci G, Livak KJ, Bi W, Strout MP, Bloomfield CD, Caligiuri MA. Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia 1998;12:1482-1489.
17. Mensink E, van de Locht A, Schattenberg A, et al. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. Br J Haematol 1998;102:768-774.
18. Suryanarayana K, Wiltrout TA, Vasquez GM, Hirsch VM, Lifson JD. Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS Res Hum Retroviruses 1998;14:183-189.
19. Morris T, Robertson B, Gallagher M. Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system. J Clin Microbiol 1996;34:2933-2936.
20. Witham PA. PCR based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Appl Environ Microbiol 1996;62:1347-1353.
21. Batt CA. Molecular diagnostics for dairy-borne pathogens. J Dairy Sci 1997;80:220-229.
22. Chen S, Yee A, Griffiths M, et al. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int J Food Microbiol 1997;35:239-250.
23. Swan DC, Tucker RA, Holloway BP, Icenogle JP. A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA. J Clin Microbiol 1997;35:886-891.
24. Brandt ME, Padhye AA, Mayer LW, Holloway BP. Utility of random amplified polymorphic DNA PCR and TaqMan automated detection in molecular identification of Aspergillus fumigatus. J Clin Microbiol 1998;36:2057-2062.
25. Desjardin LE, Chen Y, Perkins MD, Teixeira L, Cave MD, Eisenach KD. 1998 Comparison of the PE/ABD 7700 system (TaqMan) and competitive PCR for quantification of IS110 DNA in sputum during the treatment of tuberculosis. J Clin Microbiol 1998;36:1964-1968.
26. Higgins JA, Ezzell J, Hinnebusch BJ, Shipley M, Henchal EA, Ibrahim MS. 5'-Nuclease PCR assay to detect Yersinia pestis. J Clin Microbiol 1998;36:2284-2288.
27. McGoldrick A, Lowings JP, Ibata G, Sands JJ, Belak S, Paton DJ. A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan). J Virol Methods 1998;72:125-135.
28. Livak KJ, Goodsaid F. Allelic discrimination using the 5' nuclease assay. Foster City, CA: PE Applied Biosystems Bulletin, 1997.
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