PCR

PCR can identify the genes present in a sample and measure their expression, and is one of the most common techniques used in laboratories worldwide. Thistechnique is used for applications as diverse as clinical diagnosis, forensic science and paternity testing, and led to the sequencing of the first human genome.

As PCR evolved, scientists developed variations on the technique, including a form of PCR called real-time PCR (also known as quantitative PCR or qPCR). qPCR generates a fluorescent signal that corresponds to the number of copies of DNA being made as the reaction proceeds, rather than at the end of the reaction. This removes the need to quantify DNA products using time-consuming gel electrophoresis protocols.

Applied Biosystems' TaqMan qPCR is based on the most important enzyme in PCR, Taq polymerase. With an average workflow time of just two hours, Applied Biosystems' TaqMan qPCR assays exploit the 5' nuclease activity of Taq Polymerase and fluorescent hydrolysis probes to increase DNA detection specificity. It is precise, sensitive and easy to use.

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How TaqMan works

Taq polymerase is derived from bacteria that tolerate very high temperatures. This enzyme operates at an optimum temperature of 75–80°C [1]. The ability of Taq polymerase to withstand heat is critical for PCR, which requires high temperatures to separate the two strands of DNA prior to copying. Like any PCR, TaqMan requires a polymerase, a DNA template, two primers specific to the region to be amplified, and also requires a unique, sequence-specific probe.

Taq polymerase moves along the template strand, adding nucleotides to the 3’ end. As Taq polymerase extends the primers, a complementary strand of DNA is produced. Unlike the primers, the probe cannot be extended by Taq polymerase as it does not contain a free hydroxyl. Instead, on the 5’ end of the probe is a reporter dye, which generates fluorescent light proportional to the amount of DNA produced. On the 3' end of the probe is a quencher. While the probe is intact, the reporter dye is in close proximity to the quencher which as the name suggests, quenches the fluorescence emitted by the reporter dye due to fluorescence resonance energy transfer.

When Taq reaches the fluorescent probe, in addition to its polymerase activity, Taq also has an exonuclease property to partially displace and cleave the probe thus removing it from the DNA template. This separates the reporter from the quencher and generates a permanent increase in fluorescence that correlates with DNA doubling.

As well as separating the reporter dye from the quencher, cleavage of the probe also removes the probe from the target, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.

Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.

Quenchers used on TaqMan probes can vary. The most common is a non-fluorescent quencher with a minor-groove binding (MGB) molecule. This strengthens probe binding and enables TaqMan probes to be much shorter, while maintaining their high melting temperature (Tm). It is important to note that the use of an MGB moiety does affect the Tm of the probe, so the sequence cannot be used with other quencher molecules or the probe must be redesigned.  QSY quenchers are available when multiplexing 3 or more probes.  QSY is a non-fluorescent quencher that does not have an MGB moiety so the probes will tend to be longer than their MGB counterparts in order to maintain the same Tm.

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Temperature—the key to a successful experiment

qPCR depends on a series of temperature-dependent reactions, so maintaining the right temperature at the right time is key for a successful assay. In addition to the essential temperature cycles in PCR, primer pairs must have Tms within 1°C of each other.

The Tm difference between the primer and the probe is critical. The probe is the target-specific sequence that must bind to the DNA before the primers. For gene expression assays, the Tm of the probe should be about 10°C above that of the primer to enable the probe to bind to the template strand before Taq polymerase reaches it. Also important are primer annealing temperatures (Tm), which should generally be 58–60°C  and less than 50% GC content; more  could lead to inappropriate hybridization.

A typical thermal cycle profile consists of heating the reaction to 95°C to denature the double-stranded DNA, then cooling the reaction to 60°C. As the reaction cools, the TaqMan probes bind to their target sequence, followed by the forward and reverse primers.

In addition to specificity, the design of the primers and probe is crucial for qPCR to allow the probe to bind to a specific site (between the primers) before the primers themselves bind to the DNA template.

This design is key to qPCR because as soon as the primers bind, the Taq polymerase will begin the extension of the primers. If there is not a probe in the way to be cleaved, no fluorescent signal will be generated, and the efficiency of the experiment will appear artificially low. This is because even though amplicons are being generated, there would be no record of their production.

Due to the size of the typical amplicon (less than 150 base pairs), qPCR reactions have been simplified to a two-step reaction, compared with the conventional three-step process for traditional PCR. The abridged two-step thermal cycle profile combines the annealing and extension step at 60°C, making binding of the probe prior to the sequence-specific primers a critical feature of TaqMan chemistry. This thermal cycling is repeated 40 times, doubling the amount of product each time.

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Applications

Applied Biosystems’ primers and probes use a tried and tested algorithm to optimize the performance of your assay. TaqMan assays can help you answer a wide range of research questions and have been cited in more publications than any other qPCR assay product.  All assays are designed to work in PCR reactions running universal cycling conditions.   This makes it easy to run multiple experiments on the same plate or multiplex in the same well.  For more information on multiplexing refer to the TaqMan Multiplex PCR Optimization manual (MAN0010189)

One of the major uses of the TaqMan assay is to measure the expression of genes of interest. This is important in many areas of research, for example, to understand disease mechanisms by comparing the expression of different genes from healthy and diseased samples. By first creating a single-stranded DNA template from the expression transcript (mRNA), TaqMan can quantify the expression of different genes in real time.

TaqMan probes can help identify viruses and bacteria, an important function for clinical and research purposes. TaqMan has been applied to identify cases of HIV, West Nile virus, dengue fever and hepatitis [2,3,4,5,6,7]. Outside medicine, TaqMan assays are used in agriculture to quickly and reliably identify pests in plant species [8,9,10].

Applied Biosystems' allele-specific TaqMan SNP Genotyping assays are also useful for analyzing variation between samples, for example identifying the particular genetic variants associated with diseases [11,12,13]. TaqMan even detects microRNAs associated with disease, as demonstrated recently for the pregnancy disorder preeclampsia [4]. Researchers have also used TaqMan to rapidly and reliably quantify gene expression in different brain regions [14], and to detect genetic markers in brain cancer [15].

This hugely versatile technique comes in a variety of formats, and it offers advantages for all fields of research. Each assay contains everything you need to begin—target primers and an optimized sequence-specific probe—with no extra design, optimization or lengthy melt-curve analysis necessary.

You can even design your own custom TaqMan gene expression assay for use in studying your chosen gene or variant, in your chosen organism. There are over 30 species available and 1.8 million predesigned assays you can order online.

To find out more about TaqMan, check out our series of videos on real-time PCR and submit your own questions here.

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References

  1. Lawyer FC, Stoffel S, Saiki RK et al. (1993) High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5’ to 3’ exonuclease activity. PCR Meth Appl 2(4):275–287. doi: 10.1101/GR.2.4.275.
  2. Lanciotti RS, Kerst AJ, Nasci RS et al. (2000) Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. American Society for Microbiology 38(11):4066–4071. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11060069 (Accessed: 5 December 2017).
  3. Costafreda MI, Bosch A and Pintó RM (2006) Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. Appl Environ Microbiol. American Society for Microbiology 72(6):3846–3855. doi: 10.1128/AEM.02660-05.
  4. Martinez-Fierro ML, Garza-Veloz I, Gutierrez-Arteaga C et al. (2017) Circulating levels of specific members of chromosome 19 microRNA cluster are associated with preeclampsia development. Arch of Gynecol Obstet. doi: 10.1007/s00404-017-4611-6.
  5. Callahan JD, Wu SJ, Dion-Schultz A, Mangold BE et al. (2001) Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus. J Clin Microbiol. American Society for Microbiology 39(11):4119–24. doi: 10.1128/JCM.39.11.4119-4124.2001.
  6. Morris T, Robertson B and Gallagher M (1996) Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system. J Clin Microbiol34(12):2933–2936. Available at: ncbi.nlm.nih.gov/pubmed/8940425 (Accessed: 5 December 2017).
  7. Désiré N, Dehée A, Schneider V et al. (2001) Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J Clin Microbiol. American Society for Microbiology 39(4):1303–1310. doi: 10.1128/JCM.39.4.1303-1310.2001.
  8. Weller SA, Elphinstone JG, Smith NC et al. (2000) Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl Environ Microbiol66(7):2853–2858. doi: 10.1128/AEM.66.7.2853-2858.2000.
  9. Jothikumar N, Lowther JA, Henshilwood K et al. (2005) Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl Environ Microbiol. American Society for Microbiology 71(4):1870–1875. doi: 10.1128/AEM.71.4.1870-1875.2005.
  10. Qvarnstrom Y, da Silva ACA, Teem JL et al. (2010) Improved molecular detection of Angiostrongylus cantonensis in mollusks and other environmental samples with a species-specific internal transcribed spacer 1-based TaqMan assay. Appl Environ Microbiol 76(15):5287–5289. doi: 10.1128/AEM.00546-10.
  11. Kondkar AA, Azad TA, Almobarak, FA et al. (2017). Polymorphism rs10483727 in the SIX1/SIX6 gene locus is a risk factor for primary open angle glaucoma in a Saudi cohort. Genet Test Mol Biomarkers. doi: 10.1089/gtmb.2017.0159
  12. Gudz AS, Ziablitsev SV and Zaharevich GE (2017) Influence of VEGFA gene polymorphisms rs2010963 and rs699947 on clinical and laboratory indicators in diabetic retinopathy among patients with type 2 diabetes mellitus. Int J Endocrinol 13(7):471–477. doi: 10.22141/2224-0721.13.7.2017.115745.
  13. Abbasi F, Mansouri R, Gharibdoost F et al. (2017) Association study of CD226 and CD247 genes single nucleotide polymorphisms in Iranian patients with systemic sclerosis. Iran J Allergy Asthma Immunol 16(6):471–479. ISSN 1735-5249.
  14. Medhurst A, Harrison D, Read S et al. (2000) The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J Neurosci Methods 98(1):9–20.
  15. Dötsch J, Repp R, Rascher W et al. (2001) Diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas. Expert Rev Mol Diagn, Taylor & Francis, 1(2):233–238. doi: 10.1586/14737159.1.2.233.

Additional references

  • Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Nat Biotechnol. Nature Publishing Group, 11(9):1026–1030. doi: 10.1038/nbt0993-1026.
  • Wang T, Brown M (1999) mRNA quantification by real time TaqMan polymerase chain reaction: Validation and comparison with RNase protection. Anal Biochem 269(1):198–201.
  • Watson, DE, Li B (2005) TaqMan applications in genetic and molecular toxicology. Int J Toxicol 24:139–145. doi: 10.1080/10915810590948299.