Question

1. Describe the working principles behind Quantitative-PCR (P-PCR) and RNA interference (RNAi) and how they can be used to study gene expression.

Answer 1

Quantitative Polymerase chain Reaction is also called as real time PCR.qPCR actually quantifies the amount of DNA to be determined in real time.This is very much used as the method of gene expression.

Principle of real-time PCR lies in the fact that the process uses a florescent reporter whose signal strength is directly proportional to the molecules in the DNA amplification.It is one of the best analyzing technique so far.There are two kind of detection method.One is by using Taqman probe and the second method is based on the use of non-sequence specific double stranded DNA binding dye called SYBR green.

There are four steps in the RT-PCR.

1.Linear ground phase,wherein the PCR just starts,and the fluorescent signal is present in the background but is not visible.

2.This is the second phase called as the early exponential phase,wherein the fluorescent signal just start to rise about the threshold called cycle threshold.

3.This is the linear exponential phase,wherein the PCR doubles the concentration of DNA in each cycle.And this the peak of the process.

4.And the last phase is the plateau phase,which is the phase when all the substrate function lost,fluorescent signal cannot increase further as the Taq DNA polymearse comes to end.

Taqman probe is the commonly used method in RT-PCR.This is the sequence specific oligonucleotide with a fluorescent reporter at 5' and a quencher dye at 3'.When this probe is intact and does not hydrolyzed by the action of Taq DNA polymerase,fluorescent light emitted and this is absorbed by the quencher dye at the 3'.Whereas,when the probe gets hydrolyzed,there is a separation happens between the dyes at the 5' end and there will be no more quenching effect.Hence,the fluorescent light detected by the RT-PCR.

This is very much important to note that the dye signal is proportional to the amount of the product of PCR.

RNA interference: - It is the method of post translational gene silencing,where the RNA molecule actually inhibit the gene expression by the process of neutrilization on the mRNA molecule.It is a very conserved process.Only the targeted mRNA molecules are silenced after the process of translation.

The method involves the RNA degradation to the short segments of RNA calles small interfering RNA (siRNA).Following the step,unwinding of these siRNA happens due to activity of endonuclease argonote 2 (Ago2).After this unwinding,the guide strand moved to the RNA interference specificity complex (RISC) and the passenger strand get released.

This RISC then find the target mRNA.This guide stand have a complementary sequence for the target mRNA and then the endonucleotic cleavage of the mRNA happens.