Skip to main content

Precise DNA cut!

We all know that we can cut and engineer our DNA or DNA of any other organism. But, so far the methods used for DNA cutting are not precise.

Available techniques:
By the older method, the genes are added into the cell and they get inserted into the genome of the cell in a random manner. This can’t be used when our aim is to engineer the DNA at a specific site and here we can’t also replace the already existing gene with our gene.

There is homologous recombination method, but unfortunately natural recombination won’t occur in cells that easily!

There are also methods which use Zinc fingers for delivering nucleases which cuts the DNA, but, this is really hard, because nuclease can’t target every possible DNA sequence. This is costly too.

TALENs – Transcription Activator Like Effector Nucleases – cut the DNA at specific location. But, what to do, this is also costly!

Our new method
Here, we are going to use the already  existing proteins of bacteria and  RNA. The tool contains RNA sequence with the nuclease called cas9.

Here, RNA is used for recognising the site on the genome where we have to make a cut and Cas9 for cutting! The Cas9 get activated once the RNA finds exact match. It won’t get activated, even if there is a single residue difference.

If we are willing to replace the already existing sequence with new one, we have to provide a DNA template.

This is not costly, yet effective and precise!

This is a good news for all Genetic Engineers, right? Even, I’m happy about this as a GE student, then for sure this is going to be a boon for Genetic Engineers.

After all, as my professor said, Playing with genes is the real fun! J


  1. Random browsing took me here :) Nice write-ups about various biotech related stuff, I could learn some new things.

    I am not a biotech guy, but, from what I know, I would like to post some details here. As a biotech student, you may be knowing it already.

    DNA can be precisely cut using Restriction Enzymes. For example, you have mentioned TALENs, which are artificial restriction enzymes. Restriction enzymes make a cut at a specific place on both the bases of a ds-DNA, thus making some engineering possible. Restriction enzymes are available naturally and as far as I know, are not very expensive.

    What you are discussing here seem to focus more about Genome editing with engineered nucleases, which generally are artificially engineered nucleases. I know nothing about these artificially engineered nucleases, but came to know something about it from your blog.

    Have a good day!!!

  2. Actually this is not for performing invitro recombination like using enzymes like E.coR1 and ligases for cloning. This is for modifying the genome of humans or other mammals for knocking out genes which could cause diseases.

    The enzyme here mentioned is a natural one available in Bacteria - this enzyme is generally used by the bacteria for protecting itself from viral infections.

    Here we are using that enzyme in addition to that we are adding RNA sequence for recognising the sequences (where we have to make a cut).

    Hope you got it and I believe I understood the mechanism and explained it right!

    Thanks for sharing.

  3. @Kanmani: Thanks for the details. I got your point, actually, your first few lines of introduction to the topic saying that "methods available so far to cut DNA are not precise" made me write about restriction enzymes.


Post a Comment

Popular posts from this blog

Genotyping, Phenotyping, Karyotyping!

We are going to discuss here about three different typing! They are genotyping, phenotyping and karyotyping. Before learning about them let us learn what is genotype, phenotype and karyotype!

Genotype refers to the genetic make up of an organism. Generally the genes of an organism. The genotype of an organism can be represented as BB or Bb or bb based on the gene. If a person is having two recessive CFTR genes, then he will be getting cytic fibrosis. Genotype of an organism has also effect over the phenotype. Thus, genotype is representing the alleles of a gene in general. Genotyping is generally done based on PCR or hybridization.

Phenotype refers to the visible characters like structure, color and also the biochemical characters. This is based on the phenotype. Phenotyping can be done using biochemical assays.

Karyotype refers to the number of structure of chromosomes in an organism. karyotyping is done by staining and visualising the cells under microscope.

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right?

So, let me share with you some basic principle and protocol for isolating monocyte from blood sample.

For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle.

Peripheral blood sample - It is the blood sample obtained from acral areas of body (in general, it is the blood collected from ha…

Extraction of Plasmid DNA - Principle behind usage of various reagents

Have a good time :) Last time I had explained you the procedure for "electroporation" and now with those electroporated cells, you can extract the plasmid DNA.

I'm not going to share the exact protocol here, but the mechanism behind the protocol we use in our lab.

Generally, the following reagents are used for the extraction of plasmid DNA.,
1) STET buffer
4)Phenol chloroform
5) Alcohol
6) Sodium acetate

If you need the protocol, kindly mail me I'll send you the  protocol. Now, let us discuss the principle behind the usage of each of these reagents.

STET buffer - contains Sucrose which helps in maintaining osmotic pressure; Triton X which helps in cleaving the cell wall; EDTA which acts as a chelating agent; Tris HCl is used as buffer.

Lysozyme - cleaves the cell membrane and wall;

RNAase- As  the name indicates, it cleaves RNA

Phenol chloroform (buffer saturated- pH 8) - Phenol denatures proteins, Chloroform prevents oxidised phenol from bind…