Thursday, January 16, 2014

Real Time PCR (qPCR)

I don't know why, but, I really love this guy! :P Yes, he is PCR! You ask me anything, like, what technique we can use for introducing mutation? what for diagnosis? I'll answer PCR for all! :P Let me talk about him for sometime here.

PCR (Polymerase Chain Reaction) is used for amplifying any gene from a given sample using Taq polymerase, dNTPs, Primers and buffers. After PCR the amplified product or amplification is generally checked by running an Agarose gel electrophoresis. But, in case of Real Time PCR there is no need for running gels as the progress of the PCR is monitored online with the help of fluorophores. Generally SYBR Green dye, Taqman or molecular beacon probes are used for Real Time PCR. In SYBR Green method, the fluorophore binds with the double stranded DNA and produces fluorescence, and hence as the amplification increases, fluorescence increases, but the fluorophore has no specificity and hence, even if the amplified product is not the product of your interest you would find increase in fluorescence. The TaqMan and molecular beacons are probes which bind which are labeled with fluorescent dyes at one end and quencher at other end. When the fluorescent dye and the quencher are at close proximity there won’t be any fluorescence but on amplification the fluorescent dye and the quencher gets apart thus causing increase in fluorescence. These are specific to the gene amplified and hence we could monitor the amplification specifically online.

Another method is LUX (Light upon eXtension) in which the primer is labeled with fluorophore at the 3’end. The primer remains in a hair pin like structure initially, and, at that configuration it emits less fluorescence. When the primer becomes linear single strand, the fluorescence increases and when it gets extended and becomes double strand, the fluorescence increases further. As the primers are specific, the fluorescence increases only when the amplification is specific.

Melting curve analysis is generally done for the PCR products in case of LUX and SYBR Green as the fluorescence could also be due to the primer dimer. In the melting curve analysis, the product is heated thus allowing it to unwind, the melting point temperature (Tm time taken for half of the sample to unwind as single strand) is noted. The melting temperature will be different for primer dimers and the product as they differ in length. And hence, fluorescence formed could be exactly correlated only with the product after this analysis. Many of the light cyclers have inbuilt option for performing this melting curve analysis.

It has various applications; few of them are listed below:

  • Diagnosis where genes which are specific for a particular species can be targeted and amplified, if the amplification is positive then the sample could be diagnosed as infected. The specialty of qPCR is that it also gives exact severity of infection.
  • Microbiology – for comparing the differences between different species, sub species and serovars of microbes
  • Research  - for works like cloning amplification of a particular gene is essential and also for measuring expression of a particular gene qPCR can be used.
Hope, you also loved him! ;) 

Tuesday, January 14, 2014

Knock Out Mouse (KO Mouse)

Knock Out mouse is a genetically modified mouse in which a particular gene is removed or inactivated. As a particular gene (which encodes for a particular protein) is inactivated, the mouse shows changes in external features or physical and biochemical characteristics. The first recorded knock out mice was produced in 1989 for which Martin Evans and Oliver Smithies were awarded Nobel Prize.

The various reasons for producing knock out mice are

·        To understand the function of a gene
·        To understand various diseases which are due to alterations or mutations in genes
·        It gives an idea for understanding and treating various diseases
·        To develop and test various drugs produced

Some of the diseases which are widely studied using mouse models are neuronal diseases like Parkinson’s disease, Alzheimer’s disease and various types of cancer are also understood with the help of KO mouse models. Besides these, KO mice are also used for studying heart diseases, diabetes, obesity etc.

For producing knock out mouse, a DNA fragment is designed such that it contains some random sequence in place of the gene to be knocked out with flanking sequences similar to that of the sequences which are flanking the particular gene (to be knocked out) in the original genome. Moreover, the DNA fragment also contains markers to detect the effect of knock out or Embryonic stem (ES) cells are generally used for knock out, the ES cells are collected and transfected with the DNA fragment designed. Homologous recombination occurs resulting in knock out and the cells which are knocked out are transferred back into the blastocyst, then the blastocyst is placed in the uterus of a surrogate mother (mouse) which gives birth to knock out mice.

Why knock out “mouse”?

The next question that comes into our mind is “why mouse? Why not other species?”
The reasons for using mouse as a model for understanding human diseases and genes are the similarity between the genome of mouse and humans to a greater extent. And also mouse is easy to handle.

There are also limitations in producing knock out mouse. Most of the knock outs are lethal so the knocked out mouse cannot grow up and survive for a longer period. It is difficult to produce a knock out mouse as there are always possibilities for random or non homologous recombination. Producing knock out rat is difficult, and the first knock out rat was produced in 2003, 14 years after the production of KO mouse.