Monday, January 21, 2013

How to clone a GENE?


Wish you a very happy day! Let all you researches result only in success! J

You know, I’m learning cloning, wow, rhyming it is, learning – cloning!

Like this rhythm, cloning is also fun! Yes, fun, interesting! Let me share with you, what I know about cloning.

The first step of any cloning experiment would be selecting a vector. Now, I’m learning cloning using plasmid as a vector.

So, you have to choose a vector for cloning at first. Vector is nothing; it’s just a plasmid DNA into which we can incorporate our gene of interest and transform the vector into a host which will produce the protein coded by our gene of interest.

Vector need not be only p-DNA, viruses could also be used as vector. But, here, we are going to have an overview of cloning using Plasmid vector.

Okay, we had selected the vector, let us assume. What next? We have to look at our gene of interest now. We have to get the sequence of our gene of interest – it’s available in databases like NCBI.

Then, we have to isolate our gene of interest from the total genome of the organism. If it I prokaryote then you can just easily get the total genomic DNA and do a PCR (polymerase chain reaction) for amplifying your gene of interest.

If it is going to be an eukaryotic gene, then you must go in reverse. You must get the total RNA, then from that you have to isolate the mRNA, then from the mRNA you have to get your amplified gene of interest.

Before doing PCR, you must design Primer. The primer must have the restriction sequences at the end, so that the restriction sequence will also get inserted with your gene of interest at the ends.

Then after PCR, the gene f interest could be inserted into the vector just by restricting both your gene of interest and vector. They both will have complementary cohesive ends, so that they could hybridize with each other.

Of course, here there will be nick, but, we need not worry about the nick, once we transform this vector with gene of interest into our host – say E.Coli DH5 alpha, the host mechanism will take care of the nick.

E.coli DH5 alpha is a special strain of E.coli in which the defense mechanism against the foreign gene is knocked out, so that we could transform our gene into it and ask the E.coli to produce our protein, with out any problem of defense action.
This is just an overview of Cloning. There are several things to explain than this.

I’m happy that I know how to clone now! 

Happy cloning!!!

Saturday, January 5, 2013

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

Aspartame - Artificial Sweetener!

We all love sweets, isn’t it? From a cute baby who had just started feeling and enjoying the tastes to 100 year old people, love sweets! Oh, I could hear you, yes, Diabetic patients can’t enjoy sweets. But, they too love to have sweets. Even, my father is diabetic and he uses to eat sweets hiding from my mom!

Okay, what for that sweet love, you are asking? Nothing wrong in that, but, if you are crazy about the bottled drinks, then, I’m sorry, you must start worrying! L

Aspartame – is an artificial sweetener which is used in most of the bottled drinks. And, that is an approved artificial sweetener by FDA (US Food and Drug Administration – which tests and approves all food and drug items before they are marketed)

“It is approved by FDA, then, why we must worry?”, is this your next question? Let me answer you!

Aspartame is formed using two aminoacids – L-Phenyl alanine and L aspartate. This when broken down during metabolism in our body produces – phenylalanine and hence must be avoided by people who are suffering from the genetic disorder called Phenyl Ketone Urea (PKU) – these people can’t metabolise phenyl alanine further!

This Aspartame will be unstable at higher temperatures and they are stable in the pH of 4 in general. As most of the bottled drinks have the pH of 3 to 5, this is generally used in bottled drinks.

Moreover, this is preferred because this has 200% greater sweetness than the normal sucrose we use. This is enough for us right? The taste? The taste is enough, right?

Okay, what is the problem in consuming this aspartame? Only the PKU people must avoid this?

No, even normal healthy individual’s health will be affected on consuming aspartame in long run – many researches revealed this!

There are several published articles narrating the link between the aspartame consumption and Cancer! Yes, cancer, leukemia!

In 1965 Aspartame was discovered and was approved by FDA initially in 1974. Even after the approval, the safety of aspartame is being questioned by many researchers and the FDA considered the approval again!

A trick to hide the risks behind a product is generally done by showing the results of short term tests and analysis. People are misled by such short term analysis!

The metabolized products of aspartame not only include aspartate and phenyl alanine but also methanol. Methanol is toxic! But, many scientists and researches argue that methanol could be degraded by alcohol dehydrogenases. But, this degradation results in formaldehyde which will degrade DNA and proteins! This will lead to several Auto immune diseases and cancer!

And, researches reveal that men are affected more by the consumption of aspartame!
Many products like this, which are synthesized artificially might have long term effects over our health, as this is going to affect us only in long run we won’t realize this.

My mom use to advice me not to take bottled drinks and she suggests fresh juices instead. She says, if I drink bottled items, it would affect my health.

It’s true isn’t it? Aspartame or other artificial sweeteners might be the reason behind this?

I’m not sure about the worst effects of Aspartame, I just wrote this post by reading various articles and information already published.

You are sick? May be it is due to artificial sweeteners? To check this, just avoid artificial sweeteners. Check the label of everything you consume sweet! If you are fine after avoiding these artificial sweets, then, the problem is with the artificial sweeteners.

 Just try to be safe with natural sweets!