Question

What are the differences in proteins, primers, and experimental procedures for the following PCR based experiments?

a. SYBR Green qRT-PCR

b. RAPD

c. Immuno qRT-PCR

d. PCR-OLA

e. Taqman Assay

Answer 1

a. SYBR Green qRT-PCR; This is a type of real time PCR which gives the real time quantification of DNA. SYBR green dye binds at the minor groove of the double stranded DNA. When enzyme amplifies the DNA, SYBR green dye binds to the newly synthesised DNA. So the signal increases with increasing SYBR green bound to the newly formed DNA.

b. RAPD (Random Amplification of Polymorphic DNA); Its a type of PCR performed with random primers. No clue where in the genome these primers bind. So the amplification product also will be random. This technique is used with an organism whose genome is not known. This is useful to find out the phylogenetic relationship among organisms.

c. Immuno qRT-PCR; This is a powerful technique of immuno detection which combines the specificity of ELISA with signal amplification of PCR. Immuno RT-PCR depends on the use of antibody which is conjugated to an oligonucleotide and this conjugate acts as a bridge between immuno reaction and DNA amplification. Secondary antibody which has been coupled to an oligonucleotide reacts with the antigen to be checked. DNA will then be amplified.

d. PCR-OLA (polymerase chain reaction - oligonucleotide ligation assay); This method combines polymerase chain reaction with ligation assay. This distinguishes between ligation and absence of ligation of two nucleotides. Even a single nucleotide mismatch prevents them from ligation. So we can distinguish the wild type with the mutated one. This is used to find out the allele difference in a gene.

e. Taqman assay; This is a real time PCR technique which relies on two primers and and a probe. The probe is designed to bind on DNA in between two primers. Probe is made up of reporter and quencher. When these two are nearby, there will be no signals in it. When it is broken down by 5' to 3' exonuclease activity of DNA polymerase while running on DNA during polymerisation, this emits signal. The signal is proportinal to the amount of DNA poymerised.