RealTime Polymerase Chain Reaction (RealTime PCR)

In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target(1).
Figure 1. Performance characteristic of TaqMan probe
In Abbott RealTime system, Probe has been developed to suit the individual virus types. Three kinds of probe now using in Abbott RealTime system describe below ( 2, 3, 4).
  1. Linear Probe (Taqman Probe). In normal condition, fluorescence quenching occurs through random probe coiling. During the amplification step probe must bind to a complementary strand of target DNA at 60°C and in this temperature Taq polymerase using 5'-3'exonucleases activity to separating the fluorescent dye from the quencher. Probe hybridization temperature must be higher than extension temperature to ensure specificity of the reaction. TaqMan Probes can be used for discriminating genetic diversity, but probe design is less flexible. This kind of probe in Abbott, used for Hepatitis B Viral load.
  2. Partially double-stranded linear DNA probe is a fluorescent-labeled hybridization probe and a shorter complementary quencher. The 5' end of the target binding strand and the 3' end of the second strand are labeled with a fluorophore and a quencher, respectively. In the absence of target sequences, the duplex DNA formation brings fluorophore and quencher together. Binding of target nucleic acids displaces the quencher strand and leads to an increase in fluorescent signal. Partially double stranded linear probe is the unique probe, developed by Abbott for HIV-1 Viral load assay.
  3. Single Stranded Linear Probe is a kind of linear probe. In the absence of target, the probe achieves quenching through random coiling. In presence of target during amplification, the probe prefers to hybridize to the target sequence, allowing fluorescent detection without using exonuclease activity. This probe is used in various tests of Abbott molecular such as Hepatitis C Viral load, Abbott RealTime CT/NG or Abbott RealTime High Risk HPV.

References:

1. www.wikipedia.com
2. www.abbott.com
3. M. J. Espy, J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter,J. D. C. Yao, N. L. Wengenack, J. E. Rosenblatt,F. R. Cockerill III, and T. F. Smith. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin Microb Rev 2006; 19(1): 165-256.
4. Shihai Huang, John Salituro, Ning Tang, Ka-Cheung Luk John Hackett, Jr Priscilla Swanson, Gavin Cloherty, Wai-Bing Mak, John Robinson and Klara Abravaya. Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR. Nucleic Acids Res. 2007; 35(16): e101
5. Next-Generation Genome Sequencing: Towards Personalized Medicine. Edited by Michal Janitz Copyright _ 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei ISBN: 978-3-527-32090-5