Sunday, March 31, 2013

Southern Blotting / Hybridisation!

Southern blotting is used for finding specific DNA sequence from a DNA sample by using the combination of techniques like  gel electrophoresis, capillary electrophoresis and hybridization. The DNA from the gel (after gel electrophoresis)is transferred to a nitrocellulose membrane and hybridization analysis is done for finding out the sequence of our interest using a probe)

Let us discuss here the steps involved in doing Southern blotting. The steps are
1) Preparation of Sample
2) Restriction of the Sample
3) Gel Electrophoresis
4) Pre-treatment of the gel
5) Blotting
6) Hybridization

1) Preparation of Sample: (plant/animal/bacterial)

For tissue or blood samples the DNA could be obtained by treating with SDS (Sodium dodecyl Sulphate)
If we are going to have the DNA from a bacterial sample, there are separate protocols for extracting genomic DNA and pDNA. For extracting DNA from plants, CTAB (cetyltrimethyl ammonium bromide)
is used. This CTAB binds with the DNA and helps in better extraction using phenol. This CTAB is used specifically for plants, because they contain more carbohydrates than animal and bacterial cells.

2) Restriction of the Sample:

Specific restriction enzymes could be used fro restricting the sample of DNA.  Only one restriction enzyme can be used or more than one enzyme could also be used.  For restricting the sample of DNA containing 1 micro gram per microlitrethe following amount of buffer, enzyme could be used. (Choose the buffer corresponding to the enzyme you use) This volume could also be scaled up.

Volume (micro litres)
10X buffer

3) Gel Electrophoresis: 
Agarose gel electrophoresis is the next step. The restricted sample is run in the agarose gel (% of the gel depends on the sample)

4) Pre-treatment of the gel:
The gel is pre-treated with 0.25 mol/litre HCl for 30 min and also treated with an alkali.
The reasons for pre-treatment:

  • Acid treatment helps in breaking the DNA in the bands formed in the gel into smaller fragments and this also leads to little depurination!
  • Alkali treatment is done for denaturing the DNA by breaking the "H" bonds. This helps the DNA in binding easily to the membrane during the blotting. And also this helps in the hybridization with the probe.
5) Capillary Blotting:

This could be done by using nitrocellulose membrane or nylon membrane.

i) Using nitrocellulose membrane (high salt transfer)

While using nitrocellulose membrane, gel must be neutralized after the alkali treatment because at the pH above 9, there won’t be efficient binding of DNA with the nitrocellulose membrane. The neutralization is done by soaking the gel in Tris – salt buffer.

Blotting is generally done using nitrocellulose membrane. The capillary electrophoresis is set up as shown in the figure using SSC buffer (20X) (SSC contains 3mol/litre Sodium chloride and 0.3 mol/litre Sodium citrate) blotting for about 18 hours.

After the blotting or transfer is over, the membrane is washed with SSC (2X) and then dried. In this case, the DNA won’t be firmly attached to the membrane, so, the membrane could be baked at 80degree C for 2 hours which will result in semi permanent attachment of the DNA to the membrane

This same procedure followed for the uncharged nylon membrane also, till the washing and drying. After that for strong binding of DNA with the membrane, the nylon membrane is UV treated.

ii) Using a positive charged nylon membrane:
In this method, a different buffer is used and no alkali pre treatment of the gel is needed. Here, O.4 mol/litre NaOH is used as buffer and this gives immediate covalent binding of the DNA to the membrane and hence no baking or UV treatment is needed.

Advantages of nylon membrane :
i)                    Better binding of DNA, even 50bp DNA binds efficiently whereas nitrocellulose membrane efficiently binds 500bp only.
ii)                   Nylon membranes cannot be damaged easily and hybridization could be done repeatedly upto 8 or 10 times.
iii)                 No need for post blotting baking or UV treatment for +ve charged nylon membranes and has reduced loss of DNA.

Electrophoresis or electroblotting from the gel to membrane could be done instead of capillary blotting and also vacuum blotting which draws the buffer through the gel to the membrane could also be done. This reduces the time of blotting to a few hours to minutes.

Pre-hybridization step: This is done to block the areas of the membrane which donot contain DNA, to avoid random hybridization during hybridization. Generally this is done by treating the membrane in the buffer containing Salmon sperm DNA (popular choice of DNA used for blocking) for 15 minutes to 3hours depending on the membrane type.

Then actual hybridization is done by using high salt buffer with a detergent (2X SSC + 1% SDS) containing the probe DNA sequence. For increased sensitivity 10%Dextran sulphate or 8% polyethylene glycol 6000 is added to the buffer and probe mixture, this increases the cross linking between the probe and sample DNA. For increasing the specificity, the temperature at which the bonding between the probe and the sample DNA will be stable must be used; at this temperature all the other random bonding will be unstable.

Thus, we could identify the DNA fragment containing our interested sequence using Southern blotting.

Any queries? feel free to comment. 

Tuesday, March 19, 2013

Plant Bio Reactors (PBR)

You might be aware of using E.coli for producing human proteins. E.coli is a well established expression system and easy to handle. And many people are working on this E.coli (even I'm handling only E.coli in my laboratory most times)

Okay, we are fine with E.coli, and we are producing our protein of interest in that. And what is the need for an eukaryotic expression system? That thing, I explained in one of my previous posts (Clone human genes into plants!)

I explained in that post like we are expressing our human proteins and we are growing the entire plant for obtaining our protein of interest. And we need not do that all time and there is a choice of growing plant cells in bio reactors!

Yes, we do grow E.coli in bio reactors and why not the plant cells? You might be aware of this plant bio reactors and here I'm going to share something that I know about them. 

Plant bio reactors

I attended a seminar on "Advances in plant biotechnology" and as you guessed, this post is the result of that.
Okay, let me jump into the topic.

Important factors to be considered while designing a PBR are:
  • Plant cells (as well as animal cells) are shear sensitive
  • Plant cells form aggregates - and this aggregate formation sometimes helps you with your production
  • Risk of contamination

 I consider that these three things are very important in growing your plant cells in a PBR.

Shear sensitivity:
As, the plant cells are shear sensitive, we can't use the same reactor which we use for growing our microbes for growing plant cells. If you use a super fast impeller for effective mixing to eliminate the mass transfer limitations, then I'm sure your plant cells are gonna die. So, special design is needed for growing the plant cells.

So, there must be a balanced mixing such that the mass transfer is good and also the cells should
not lyse!

Aggregate formation:
Plant cells also form aggregates like bacterial cells. We don't prefer aggregate formation in case of bacterial cells. But, in plant cells, depending on the species, this aggregate formation could improve our productivity.
On the other hand, this aggregate formation causes mass transfer limitations thus affecting your productivity.

"???" You feel like "oh God"? And your question is "whether to have aggregates in plant cell reactors or not?"

Let me answer you. You need to have aggregates if it benefits you with your productivity but the same time, the mass transfer must also be good. For having the two things in balance, we have to maintain a "particular aggregate size", so that, our productivity will be fine in both the aspects like mass transfer is good and aggregate formation improving the productivity.

Then, the next question may be "how to maintain the optimum aggregate size?"

The optimum aggregate size could be maintained by using immobilization technique. You could immobilize your cells i.e. the optimum aggregate size would be maintained using immobilized plant cells.

Risk of contamination:
Comparing the bacterial cells, plant cells have a very high risk of contamination. Even when we grow them for producing callus, there is a lot of contamination risk especially fungal infection.

So, growing them in a reactor, that will be difficult job especially in case of maintaining the sterile environment. Most times, continuous reactors are not used for bacterial cultures as it has high risk of contamination comparing the batch and fed batch. I had never seen or done continuous culture of bacteria in our lab, normally we don't do continuous culture as it goes for more than a day and also it is difficult to maintain sterility. And, even when on of my seniors wanted to try continuous culturing, we were all saying "no you better try batch instead" fearing that everything may get spoiled due to contamination.

But, if high sterility is maintained, you can do continuous culturing of plant cells also.

Hope you understood what i explained, got doubts? Comment or e-mail, will answer you as soon as possible.

Sunday, March 3, 2013

Tuberculosis Diagnosis!

Hi, Today I'm going to share something about TB! TB - Tuberculosis is generally caused by Mycobacterium tuberculosis. The most common symptom of TB is "non -stop" cough!

They say, many people in India are "carrier" of the bacteria, but not the disease! Oh God! if you are in India, better go and check for this bacteria! The possibility of the spread of the disease could be due to unhygienic conditions!

You spit in public places? you sneeze? cough? No problem, you can sneeze or cough, but be careful! Careful coughing? careful sneezing? yes, you might spread the population this mycobacterium you are carrying (you need not have TB), so, be careful in sneezing and coughing!

Okay, let us come to the point, I started typing this with a view of giving an overview about "the diagnosis of Tuberculosis".

The most common test used for checking TB is "Tuberculin Skin Test". Let me explain you how this is done!

Tuberculin Skin Test!
Let us first look into the principle behind this test. When a person is infected with Mycobacterium tuberculosis, then, he will be carrying antibodies against it. When he/she is injected with the antigen from this bacteria, he will produce an induration (hard bulge in the place where the antigen is injected).  This could be found after sufficient time like 72 hours.This is used for diagnosing TB! In this case, false positive results are obtained when a person tested for the second time using this method.

Antigen? What is the antigen used here? Is that your question? It is the protein which triggers the immune response extracted from the mycobacterium sp.

 Do you think this is enough for diagnosing TB? "Okay, what else one could do for diagnosing TB?" When my professor asked this question in class, I replied "ELISA".

ELISA - Enzyme Linked Immuno Sorbent Assay: Here the blood sample from the patient must be collected and checked for antigen antibody interaction in the well plate. Here we must have the antibody available in our lab. If there is antigen antibody interaction the ELISA produces colour else it won't!

"Okay fine. What else you could do for diagnosing TB?", when my professor questioned further, I replied, "PCR"! He was happy atlast that I had answered as he expected!

PCR: How PCR could be done for diagnosing TB? Mycobacterium genome sequence is available online in the databases and one could design primers based on that and when PCR works, then the person is having TB, else not! Real Time PCR is generally preferred than the normal PCR for avoiding errors.

The advantage of PCR is that one could also say the severity of the disease and dosage of the antibiotic could be given accordingly.

After all, the dosage must be very correct and the antibiotic treatment should not be dropped in the middle to avoid development "multi drug resistant" species!

I had shared whatever I understood as an overview, any doubts? mistakes? suggestions? Feel free to comment!