It is based on PCR technology, which is used to amplify and simultaneously detect or quantify a targeted DNA molecule on a real-time basis. Reverse transcriptase real-time PCR formats can detect and quantify RNA molecules of the test organism in the sample on a real-time basis.

It uses a different thermocycler than the conventional PCR. It is very expensive, 5-10 times more than the cost of conventional PCR (Fig. 1).

Fig1. Real time PCR. Source: Department of Microbiology, JIPMER, Puducherry (with permission).

Advantages:

 Real-time PCR has many advantages over a conventional PCR, such as:

- Quantitative: rt-PCR can quantitate the DNA or RNA present in the specimen; hence can be used for monitoring the disease progression in response to treatment, e.g. viral load monitoring in HIV or hepatitis B viral infection

- Takes less time: In rt-PCR, the amplification can be visualized simultaneously during the process of amplification unlike the conventional PCR where there is an extra-step of gel electrophoresis to detect the amplicons

- Contamination rate is extremely less

- Sensitivity and specificity of rt-PCR assays are much more than the conventional PCR.

Detection of amplification products of real-time PCR:

 The detection of amplified nucleic acid in a real-time PCR reaction is carried out by using a variety of fluorogenic molecules which may be either nonspecific or specific.

- Nonspecific methods: They use SYBR green dye that stains any nucleic acid nonspecifically

- Specific methods: They use fluorescent labeled oligonucleotide probe which binds (i.e. hybridizes) only to a particular region of amplified nucleic acid. Three types of hybridization probes are commonly used:

* TaqMan or hydrolysis prob

* Molecular beacon

* Fluorescence resonance energy transfer (FRET) probe.

 Post-amplification melting curve analysis is used for quantitation of the nucleic acid load. 

مواضيع ذات صلة

الإضعاف :التوقف المبكر للاستنساخ (Attenuation: the premature termination of transcription)

كابح التربتوفان tryptophan repressor

Gel Electrophoresis Assay of Eukaryotic Replication Origins

Constancy of the E. coli DNA Elongation Rate

Bidirectional Replication from E. coli Origins

أوبرون اللاكتوز lac operon

اكتشاف الأوبرون

مثبطات تخليق البروتين .... وسيلة للعلاج

تحوير سلاسل الأحماض الأمينية الجانبية للبروتين (Amino acids side chains modifications)

تحوير التركيب البروتيني ثلاثي الأبعاد (Three dimensional structure modifications)

Error and Damage Correction

فروقات بدء الترجمة بين بدائية وحقيقية النواة