Thursday, December 20, 2012

GM Mosquitoes #2

In the previous post, GM Mosquitoes #1  we had seen about the GM mosquitoes used for curing malaria. Now, we are gonna see about the GM mosquitoes designed for eradicating mosquitoes population!

Aedes aegypti is the mosquito species which spreads Dengue. Nowadays, people are suffering severely because of this Dengue. In most cases it just leaves with mild fever. But, in some cases, Dengue is deadly! So, to get rid of this great dengue nuisance, scientists had found a solution - GM mosquitoes!

We had seen that in the case of eradication of malaria by GM mosquitoes that scientists had made the mosquitoes resistant to malaria causing protist, but, here in this case of dengue the genetic modification is different.

Dengue!
In the case of dengue, male mosquitoes are made sterile and when they mate with the wild type female mosquitoes, their young ones are made unviable i.e they die before reaching adult stage. So, this reduces the population of mosquitoes by the death of new born young mosquitoes. (they use male ones as they don't bite and feed only plant products)

How the sterile males are made?

  • In one case, they say they are injecting an artificial gene into the male mosquitoes which makes them sterile.
  • In the other case, the male mosquitoes are exposed to gamma rays which causes mutation and makes the males sterile!
Positives!
If this works out, the number of dengue victims will sharply come down. Moreover, you won't be bitten much!

Note: there is no medicine available for curing dengue.

Drawbacks!
  • Think, how harder it would be to separate male from female mosquitoes? This is the major disadvantage. :'(
  • Sudden elimination of one species may suddenly pave way for something worse ever! :(
  • If something goes wrong with this gamma ray mutation, even deadly species can come out! :O
Know this too!
  • Reports and articles I read says that already they had released these GM mosquitoes in the cayman islands.
  • Oxitec is the biotechnology company which is working on the production of this GM mosquitoes.
  • India (where i'm from) is also planning to use this technique for eradicating mosquitoes and reduce the number of dengue victims. 
Ethics!
After all, the Government or concerned authorities before releasing the GM mosquitoes into the environment, must inform the people living over there. Must get their feed backs! 

Releasing the GM mosquitoes with out prior information, won't be fine! This may lead to various ethical problems!

Just imagine!
They use males as they don't bite us (if they are biting us, then they won't try this at all, because we are always cautious that we would be affected) what if they affect plants? You  see, not a single mosquito is going to sit over a plant, when many mosquitoes sit and feed from plants, is it possible that they might affect the plants. Inturn, when we feed those plants, we will be getting affected?

Just I imagined this last paragraph, It's silly?  :)

Tuesday, December 18, 2012

GM Mosquitoes! #1

You are troubled at the mid-night? Your days are affected due to the lack of sound sleep? I can understand; The "mosquitoes", they are responsible for all this. We can bear with them if they are just biting and sucking a drop of blood, but, never, when they are injecting us with something like a "doctor", that too, which is never going to cure the disease like doctor's injection, but, going to give us disease.

Mosquito bats, liquids :( even with the help of these things, we are getting malaria, dengue, chikungunya :( :(

Scientists had found a solution for this greatest problem of our night!(nowadays daytime too)

GM mosquitoes, these genetically modified species are resistant to diseases like malaria. 

MALARIA!
How malaria generally spreads!
As we all know, its because of mosquitoes, especially the female Anopheles mosquito which gets the virus when it sucks blood from an infected animal/human.

Half of the life cycle of the Plasmodium species (the malaria causing protist) happens in the  mosquitoes. So, to prevent the spread of Malaria (or any other mosquito spread disease) we must prevent the mosquito from getting affected by the disease.

HOW Genetic Modification done?
First, the mosquito should not get affected by the malarial protist i.e. it must be resistant against the plasmodium. When this is possible,  mosquitoes are not affected by plasmodium, thus humans are also not affected.

In the case of malaria: The gene responsible for fighting against malaria must be triggered. The gene is responsible for the production of a protein (they say SM1 peptide). This protein when produced, fights against malaria, i.e, the mosquito will become resistant to Malaria!

Drawbacks:
  • These GM Mosquitoes are weak, thus, reducing their chance of survival.
In the begining of the experiments conducted in the lab, there was 50/50 chances for GM and wild.
After 9 egg laying cycles they found this incresed to 70/30 for GM and wild.

You may wonder, here, GM has good surviving rate! yes, This is in the presence of malarial infection, but, when there is no malarial infection at all (i.e. the mosquitoes suck blood from healthy individuals only), the GM mosquitoes fail to outsurvive the wild type.

  • You know, these microbes are smart! 
There is every possiblity for resistance development. These Plasmodium's could modify themselves to infect the mosquitoes, thus, leading to the evolution of a new plasmodium speices which would be more powerful than the previously existing types. And, we can't control these new protists with existing medicines or treatments.

We can look about control of Dengue using GM mosquitoes in the next post very soon.

Read more about this @ 



Thursday, December 6, 2012

Cot Value Analysis

Hi,
Wish you a good time :) Molecular biology! I was taught about "cot value analysis" in my molecular biology class. Here, you could read something, which I know about cot value.

Cot (C not (zero) t) value analysis:
This analysis uses the DNA denaturation and renaturation kinetics. This is used for measuring the repetitive DNA sequence in a given DNA sample. This analysis has application like separating repetitive sequences from mixture of DNA sample. Okay, let us discuss the procedure now.

Note:
Cot value = (C zero) * (Time) * (constant representing the effect of cation in the buffer on renaturation)
where,
c zero -> DNA concentration (mol/l)
T - Renaturation time (sec)

Procedure:
  1. Denature the DNA sample by heating
  2. Cool and renature the sample, note renaturing extent (as cot value) at particular temperatures until a particular cot value is reached.
  3. Now, dilute this sample with sodium phosphate buffer- SPB (0.03M - at this buffer concentration the DNA bind to hydroxyl apatite properly)
  4. Bind this buffer solution with DNA to hydroxylapatite(HAP) column.
  5. 0.12M SPB is now used for eluting Single stranded DNA from HAP; Collect the Single stranded DNA.
  6. 0.50M SPB is then used for eluting double stranded DNA which is also collected.
  7. Volumes of Single and double stranded DNA collected is measured.
  8. 0.9ml of the centrifuged single stranded DNA is mixed with 0.1 ml of  10N KOH. This addition of KOH denatures the double stranded DNA (if present). Do this to the double stranded DNA sample also.
  9. Absorption values A260 (adjusted for light scatter at 360nm) are obtained for both the samples prepared (mentioned in the previous point)
Percentage of single stranded DNA could be calculated with the help of the following formula:

[(Vss x Ass) x 100] ÷ [(Vss x Ass) + (Vds x Ads)] = % ssDNA
where,
Vss = total volume of single-strand fraction, 
Vds = total volume of double-strand fraction, 
Ass = A260(adjusted for light scatter) for the KOH-denatured single-strand fraction,
Ads = A260 (adjusted for light scatter) for the KOH denatured double-strand fraction.

Background:
  • The rate of renaturation of repetitive sequences will be high comparing the rate of renaturation of non repetitive or less repetitive sequences.
  • This is because, the possibility of a repetitive sequence to find it's complementary strand is greater than that of a non repetitive strand. (Hope you got it)
For detailed explanation refer "here"

Got any doubts? Kindly comment I'll try to answer.

Bye, Take CARE! :) 



Sunday, November 25, 2012

MAGNETS - a cure for CANCER!

Cancer!As you may know,  cancer is uncontrolled multiplication of cells. There are several reasons for this uncontrolled multiplication of  cells like mutations due to UV exposure or exposure to certain chemicals, even genetic defects, excessive alcohol consumption or smoking (kindly, avoid smoking or alcohol consumption). Still there are several reasons you could quote as "cancer causing".

There are different types of cancer based on the part of the body affected like lung cancer, lung cancer, blood cancer etc.,

Huge number of people all over the world are suffering from cancer, it's a deadly disease, it has no preventive measures and moreover, there is no real cure for cancer, only treatment methods are available!

Here, we are going to discuss about the possibility of using magnets in curing cancer.

When I read about usage of super magnets in treating breast cancer just by hanging the super magnet in a necklace which hangs over the breast of the patients (!), wow, how great this is! With out undergoing any painful surgeries for removing the breasts , how great this! Just by hanging a super magnet around your neck, you could treat your cancer! This is based on magnetic spirals, let us discuss this here,

Magnetic spirals!

When a super magnet is hung around your neck, it influences the whole body. This is based on the concept of "spirals" - Spirals are found through out the universe, from stars to DNA!

The basics behind this is, cancer cells have different electromagnetic potential and the healthy cells have different electromagnetic potential!

The basics is that the spirals produced by the magnets and the cancer cells are in different scales.

Super magnets produce spirals on large scale than the cancer cells. This spirals produced by the super magnets dominates the spirals produced by the cancer cells and at the end, it kills the cancer cells.

It is amazing, isn't it???

-------------

Then, another method, by combining nanotechnology and magnet in cancer treatment is also developed recently. Let us discuss that too.


Magnets! Nano particles! Iron oxide!

How magnets could be used?

The cell surface has receptors, which binds specifically with signalling molecules. Scientists had used iron oxide as a signalling molecule.

These iron oxide particles were made to bind specifically with the receptors on the surface of cancer cells, not with the receptors (particularly called death receptors) on normal cells!

When strong magnetic field is applied, these cancer cells bound with iron oxide come together and this causes apoptosis (cell death)

What is  the advantage of using this method?

This targets only the cancer cells, with out affecting the normal cells. The already existing techniques  causes side effects as it affects the healthy cells also.
-------------------------------------------------------------------------------------

I just shared what i understood by reading some papers and articles.
These are the links from where I read and summarized the above post, you could read more here,

http://news.softpedia.com/news/Cancer-Treatment-Based-on-Magnets-May-Be-the-Cure-We-ve-Been-Looking-For-298205.shtml

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002267/

http://www.godlikeproductions.com/forum1/message710345/pg1

http://www.drbakstmagnetics.com/index.php?option=com_content&task=view&id=80&Itemid=85


The concepts which I explained here may have some errors, but I took much care to provide the correct details.


Thursday, November 22, 2012

Perl Programming #5

Hi,
Didn't read the previous perl posts? Go here, perl #1 #2 #3  #4)

In the previous post of perl programming, we learnt how to search an aminoacid in a protein sequence. Now, we are going to look at a program by which the "start and stop codons" in a protein sequence could be easily identified. Here, we go,

Program to identify start and stop codons easily: 

print "enter a file containing Dna sequence\n";
$dnafile=<>;
chomp($dnafile);
unless(open(DNAFILENAME,$dnafile))
{
print "file not found\n";
exit;
}
$dna=<DNAFILENAME>;
$dna=join("",$dna);
$dna=~s/\s//g;
$dna=uc($dna);
$dna=~tr/T/U/;
print "The mrna is:\t",$dna,"\n";
my(%code)=
(
'AUG'=>'Start',
'UGA'=>'Stop',
'UAA'=>'Stop',
'UAG'=>'Stop',
);
for($i=0;$i<(length($dna)-2);$i+=3)
{
$codon=substr($dna,$i,3);
if($codon=~AUG)
{
$out.=$code{$codon};
}
elsif($codon=~UAG)
{
$out.=$code{$codon};
}
elsif($codon=~UAA)
{
$out.=$code{$codon};
}
else
{
$out.=$codon;
}
}
$out=~tr/U/T/;
print "the dna seq is\t",$out;
exit; 


When you run the above program, you would get the result just like this:

PERL
Happy perl-ing, Any doubts, just comment, I'll try to clear.
Bye. :)

Thursday, November 15, 2012

Summer Research Fellowship by IAS

Hi,
This is semester exam time! All the very best for the people who are gonna write their exams.
Are you interested very much in science? Looking for a fellowship? This is exclusively for Indian students - not for foreign students! (Sorry).



This is not only for biology students, but, also for Physics, Chemistry, Mathematics, Engineering and earth and planetary science students.

Then, this is a great chance for you. Apply for the Indian Academy of Sciences  (IAS)- Summer Research Fellowship Programme  - You could find all the details "here"

You could Register here and the recommendation letter format could be found  here !

This is for both students and teachers. While registering online, you might get doubts like, "where to select the list of guides" -

The list of guides is provided in the application form - as detail number "14"
Scroll down the application, you could find the link for selecting the guides. You can check the details like specialization of the guide, availability of accommodation etc.,

You are allowed to choose a maximum of 6 guides.

I hope this fellowship would be very useful for a successful research career. Apply soon,

The last date for applying  is December 31st. 

Any doubts? queries? Kindly comment, I'm happy to help you.

Have a great day! Bye!

* This post is written just for making the students aware of the fellowship. 

Friday, November 9, 2012

Perl programming #4

Hi,
We started learning perl programs in our last post. For viewing previous posts, use the links ( perl #1 #2
 #3 )


Today I'm gonna give you a program for searching a specific amino acid in a protein sequence.You can easily understand the logic by yourself. Here we go,

Program for searching an amino acid in a protein sequence:


print "enter a protein file\n";
$proteinfile=<>;
chomp($proteinfile);
unless(open(PROTEINFILE,$proteinfile))
{
print "file not found\n";
exit;
}
$protein=<PROTEINFILE>;
$protein=join("",$protein);
$protein=~s/\s//g;
$protein=uc($protein);
print "The sequence is",$protein,"\n";
$a=A;
$position=0;
for($i=0;$i<length($protein);$i=$position+1)
{
$position=index($protein,$a,$i);
if($position=~-1)
{
last;
}
print $position+1,"\t";
}
print "- the position(s) in which alanine is found\n";
exit;

The output for the above program will be like this. Try executing! 


Happy programming! :) :) 

Wednesday, November 7, 2012

Perl programming #3

Hi,
You read the first two posts about perl? Missed? Don't worry, go and read here ( perl #1 #2 )

From today, we can learn few programs that would help biology students! Ya, as a biotechnology student I use perl programs for dealing with DNA , RNA and Proteins.

So, I know just something about perl (very limited) for processing sequences! Okay, let's learn some basic programs.

Program to convert DNA sequence to protein sequence:

print "enter a dna sequence file\n";
$dnafile=<>;
chomp($dnafile);
unless(open(DNAFILENAME,$dnafile))
{
print "file not found\n";
}
$dnafile=<DNAFILENAME>;
$dna=join("",$dnafile);
$dna=~s/\s//g;
$dna=uc($dna);
print "DNA seq. is\n",$dna;
$dna=~tr/T/U/;
print "mRNA seq. is\n", $dna,"\n";
my(%genetic_code)=
(
'UUU'=>'F',
'UUC'=>'F',
'UUG'=>'L',
'UUA'=>'L',
'UCU'=>'L',
'UCC'=>'S',
'UCG'=>'S',
'UCA'=>'S',
'UGU'=>'C',
'UGC'=>'C',
'UGG'=>'T',
'UGA'=>'Stop',
'UAU'=>'Y',
'UAC'=>'Y',
'UAG'=>'Stop',
'UAA'=>'Stop',
'CUU'=>'L',
'CUC'=>'L',
'CUG'=>'L',
'CUA'=>'L',
'CCU'=>'P',
'CCC'=>'P',
'CCG'=>'P',
'CCA'=>'P',
'CGU'=>'A',
'CGC'=>'A',
'CGG'=>'A',
'CGA'=>'A',
'CAU'=>'H',
'CAC'=>'H',
'CAG'=>'Q',
'CAA'=>'Q',
'GUU'=>'V',
'GUC'=>'V',
'GUG'=>'V',
'GUA'=>'V',
'GCU'=>'A',
'GCC'=>'A',
'GCG'=>'A',
'GCA'=>'A',
'GGU'=>'G',
'GGC'=>'G',
'GGG'=>'G',
'GGA'=>'G',
'GAU'=>'A',
'GAC'=>'A',
'GAG'=>'E',
'GAA'=>'E',
'AUU'=>'I',
'AUC'=>'I',
'AUG'=>'I',
'AUA'=>'I',
'ACU'=>'T',
'ACC'=>'T',
'ACG'=>'T',
'ACA'=>'T',
'AGU'=>'S',
'AGC'=>'S',
'AGG'=>'R',
'AGA'=>'R',
'AAU'=>'N',
'AAC'=>'N',
'AAG'=>'K',
'AAA'=>'K',
);
for($i=0;$i<(length($dna)-2);$i+=3)
{
$codon=substr($dna,$i,3);
$protein.=$genetic_code{$codon};
}
print "the protein sequence is\n",$protein,"\n";
open (PROTEIN, ">protein.txt");
print PROTEIN $protein;
close PROTEIN;
exit;

O/P of the above perl code
"Hashing" here we are using "Genetic code" where we specified the one letter aminoacid code for the codon

"length" this command is used in the perl for finding the number of letters in the pattern
"substr" is used for getting a substring from the given string. You can specify the string from which the substring must be taken and the number of letters of the string that must be taken as substring can also be selected.

$codon=substr($dna,$i,3);

Here, we are selecting the substring from the main string stored in "$dna" and as we are going to take as codon, so, we are taking 3 letters here as substring.

In the following line,
 
 $protein.=$genetic_code{$codon};

we are converting the three letter codon into one letter amino acid code and saving it in $protein. 
We are using ".= " which appends the value when each time the for loop is executed, so that, we get the complete aminoacid sequence, rather than a single amino acid - check with out giving the "dot", you could understand it better. 

In the following lines, we are saving the output sequence in a text file, we are creating the file protein.txt. In the next line, we are printing the output in the created file, in the next line, we  are closing the file.

open (PROTEIN, ">protein.txt");
print PROTEIN $protein;
close PROTEIN;

I explained in my own way, hope i explained in a better way.
Got the concept? Any doubts??? Then, comment.
 

Tuesday, November 6, 2012

Perl programming #2

Hi,

Yesterday we learnt about installing perl in windows (Didn't read yesterday's post? check here Perl programming #1 )and how to “print” using perl!
Today, let’s start with the basics, how to get input using perl?


To get a text file from user using perl:
Just it is very simple, type as follows,

$variable=<STDIN>;

This will get the file name. This also works in perl,

$variable=<>;

No need to give STDIN! Just the open and closed angular brackets are itself considered as standard input.

Then, always give chomp after the standard input, this chomp is nothing but “chop” which chops the “enter” à new line character i.e., whenever you give an input, you press “enter” which will be considered as a new line character. In order to remove that new line character, chomp must be given.

How to use chomp? Use as follows,

$variable=<>;
chomp($variable);

Simple, this chops the new line character from your input.
Let us look at sample program for getting a text file and a word or pattern.
Program for getting a DNAFILE and printing the file content: (sample.pl)

print "enter a dna sequence file\n";

$dnafile=<>;
chomp($dnafile);
unless(open(DNAFILENAME,$dnafile))
{
print "file not found\n";
}
$dnafile=<DNAFILENAME>;
$dna=join("",$dnafile);
$dna=~s/\s//g;
$dna=uc($dna);

print "DNA seq. is\n",$dna;
close(DNAFILENAME);
exit;


O/P of the program sample.pl

Here, DNAFILENAME is the file handler. Here, we used unless for checking, whether the given file name exists or not.

For opening the file, we can give just  as,

open(DNAFILENAME,$dnafile);

don’t forget the semicolon.

Don’t forget to give the line,

 $dnafile=<DNAFILENAME>;

This assigns the content of the given dnafile to “$dnafile”
With out assigning this if you are just proceeding, you will get the output printed as the “file name” and not the file content.

“join” command is given for joining the spaces in the file. This helps when you are working with DNA and protein sequences.

$dna=~s/\s//g; This is also given for removing the spaces in the file

$dna=uc($dna); This changes the content of the file into upper case(UC)

“close” is used for closing the file,

print – It prints everything given within double quotes, even \n must be given within quotes, but, the variables like $dnafile must not be given within the quotes. Check the syntax given in the program.

Program for getting a pattern or word from the user and printing it (sample1.pl)


print "enter a pattern\n";
$pattern=<>;
chomp($pattern);
$pattern=join("",$pattern);
$pattern=~s/\s//g;
$pattern=uc($pattern);
print "Pattern is\n",$pattern;
exit;


O/P of the program sample1.pl

Here, no file handlers are used. Once you try executing the programs, you could easily understand!

Try printing the output, after using “join” and the line “$dna=~s/\s//g” for knowing their difference. Here “g” stands for global i.e., removes the space globally.

Any doubts? Kindly comment. I’ll try to clear your doubts.

:) Happy perl-ing :)



Monday, November 5, 2012

Perl programming #1

Perl is a programming language. This is a powerful language which has features similar "C". This is generally used for text processing. "Perl" is not an acronym officially, but, it has got backronyms(may be invented with a false etymology)  in use such as "Practical Extraction and Reporting Language".

In Biotechnology, this Perl is used generally for processing text data of DNA sequences and converting them to mRNA or searching for a specific pattern in a protein sequence etc.,

I'm gonna write a series "Perl programming #1, #2, #3 etc.. with simple programs and explanations. Wanna learn perl with me? Let's begin from today. I'm sure, atleast you will get to know the Basics of perl. You can learn a programming language, of course, free of cost, with in a weak!!!

What you need for perl programming?

PERL and UNIX:
If you work in UNIX, you need not install perl, just  go to terminal, and you could create a perl file just by typing as follows,

vi filename.pl

This will open a perl file, in which you can type your code.

save the file, then, type

./filename.pl 

for compiling. This will show you the errors (if any) else, the code will be executed.

PERL AND WINDOWS:

Windows users must install perl as it doesn't have perl installed by default.Use this link (http://www.activestate.com/activeperl/downloads) for downloading "active perl" based on the version of windows your working with i.e, windows xp, windows 7, download accordingly.


Download perl according to the O.S

Just download, save, run. It's very simple to install. (But, check with the O.S. and bit of the o.s. windows has 32 bit, 64 bit... check that first before downloading)

Then, go to command line,
check the directory, where you are.
for creating a perl file, type as follows,

perl >>filename.pl


command prompt - creating perl file




































This will create a file with the name  "filename.pl"

Now, "close" the command line, and, go to the folder containing your perl file, right click it, click the option "edit" and it will open like a text editor where you can type your perl code and "save".

then, come again to the command line and execute by typing,

perl filename.pl

This will show you the errors else the program will be executed.

Let us start with a simple code

just a print statement:

type as follows:

print "hello";

this will give the output as

hello

This is enough for today, let's learn the next chapter by tomorrow! :)

Happy perl -ing  :D :)

Friday, November 2, 2012

Cellular computing - Molecular computing - DNA computing

Hi,
DNA computing, this is a topic in which my professor asked me and my friend  to give a lecture. We gave our best and here you can find a summary of our lecture.

DNA computing: It's computing with the use of DNA, in stead of the normal microprocessors used.

Why DNA or cells or biological molecules for computation?
I could give you three major reasons:
1) Large storage capacity
2) High parallelism
3) Speed

Large Storage capacity: DNA could store large information comparing the normal storage devices used like CDs. If we store the information in DNA in CDs and arranga it like a thread. That thread could round the Earth by 374 times!!!! Can you imagine the capcity of DNA now?

High parallelism:  DNA could process in several ways parellely.

Speed:  All biological reactions happen with in seconds. So, using biological molecules can increase the speed of computing to a greater extent.

Okay, but, how biological molecules could be used for computing?
The basics behind the computing is the usage of logic gates.

AND , OR gates are used. Cells can also behave as logic gates.

This is possible by a technique called CID - Chemically Inducible Dimerization.

This tool takes advantage of natural biological mechanisms that
bring together two proteins into a complex in the presence of an
enzyme
• FRP and FKBP joined by rapamycin.GAI and GID1 by gibberellins.
• OR gate, FRB and GAI were bound together at the cell membrane,
while FKBP and GID1 were bound together floating freely in
the cell. Adding either rapamycin, gibberellins, or both to cells
brought the freely floating complex to the one at the cellular
membrane, linking up the matching proteins and triggering the
output signal.
• AND gate, the researchers placed just GAI at the cell membrane,
with just FRB and complexes of FKBP and GID1 free-floating in the
cell. This system required all four proteins to link up to produce
membrane ruffling, which wouldn't occur without both input
chemicals.

This look great isn't it?

DISADVANTAGES of this DNA computing:
• Just newly developing
• Some problem regarding incorporating the
circuit into the host
• Need a manual control

I had a great time during the lecture session with my friend Priya :)
We confused our class mates as much as possible :P :P

Any doubts? kindly comment, I'll try to answer. Bye.. Take care!

Viva voce #3

Hi,
During the last practical exams I shared my experience.(Read here) I shared certain questions with answers which the examiner asked me during the practicals. Even this time, I'm going to share a few questions and answers with you. Here we go,

This time I had 2 lab exam (Practicals), number one - Bio informatics and two - Molecular biology.
Let us start with the Bio informatics lab.

Bio informatics: 

In this Bio info practicals, we were provided with two exercises - one must be done using various bio info tools such as Clustal W, T-coffee, BLAST P, BLAST-N, PHYLIP etc.,
The other exercise is PERL programming! :( :'( 
The first exercise using tools is very simple, but, I find the programming, let it be any programming, it's very difficult :'( Even in the model practicals, I couldn't debug the program :(
So, I worked really hard to learn the PERL! I was working with my lap for a whole day. And atlast, I got good confidence that I could write my own program! :)
On the exam day, I got output for my program with out any errors :) 

And the viva voce, that was very simple :) I scored 9/10 :)

Okay, let us see some of the questions that examiner asked me. 

1) Question: What are the tools you are using here for Multiple sequence alignment?
Answer: I used CLUSTAL W & T-COFFEE for aligning the given 8 protein sequences. 

2)   Question: What is a phylogram?
Answer: It is a phylogenetic tree which can be drawn using both CLUSTAL W & T-COFFEE and the distance in the tree shows the evolutionary relationship between the sequences.

3) Question: What do you mean by distance?
Answer: Distance in the phylogenetic trees are calculated based on the differences between the sequences. i.e., distance shows how much the sequences are different.

4) Question: I'm giving you a protein sequence which contains mutation, and I'm telling you the function of that protein sequence, how will you identify the region of mutation?
Answer: It's very simple, we have to get the protein sequence with the mentioned function from any of the protein data bases and the we have to compare the mutated sequence with that original sequence, now, we can get the mutated region!

The questions were very simple and answered them right :) 

Now, let us discuss the molecular biology viva voce questions:

Experiment I got was "Linearise the given pDNA at E.CoR1 site"


1) Question: Procedure for Restriction digestion of pDNA?
Answer: pDNA has Multiple Cloning Site, which has several restriction sites. These restriction sites have specific sequences which are not repeated elsewhere in the pDNA. So, we have to choose a restrictions site and a corresponding restriction enzyme with corresponding buffer. We have to take 1 micro gram of DNA in an eppendorf along with 1 micro liter of buffer. To this 7 micro liter of nano pure water must be added.The enzyme must be added atlast (1 micro liter - 10 units). This sample must be incubated or digested at 37 degree celsius for an hour. Then, this sample can be used and Agarose gel electrophoresis must be performed.

2) Question: What is the site you are using here for restriction?
Answer: E.coR1

3) Question: Whether there will be only one E.coR1 site?
Answer: Yes, because, the main objective of our experiment is to linearise, and as I explained earlier there will be only one restriction E.coR1 site, If there are more than one E.coR1 site, then it would lead to fragmentation of DNA.

4) Question: Whether the linearised and the non linearised plasmid will move the same in the agarose gel?
Answer: No, the non linearised, i.e, circular DNA will move faster through the pores due to the circular shape, while, the linearised one moves slower.

That's it Viva over, answered all the questions :) :)

Hoping for "s" grade (90 to 100%) as maximum and "A" grade(80 to 90 %) as minimum.

Got any more questions regarding this? Comment then. I'll try answering you :)
Wish you a good day!

Agarose gel Electrophoresis with principle

Hi,
Agarose gel electrophoresis
In the previous post I explained how to quantify DNA, and now we can find the molecular weight of that extracted DNA.

But, how? Simple, you need to run an agarose gel!

Preparation of agarose gel:

Agarose gel must be prepared based on the DNA we are going to run. For example: if you are going to run Genomic DNA, you must use 0.6%gel, for plasmid DNA, you must use 0.8% gel, for PCR(Polymerase chain reaction) sample, you must use 1.2% gel.

You may ask, why three different % of gels? The answer for your question is:
Genomic DNA will be bulkier than pDNA - so, we must use a gel with less percentage, so that the pore size will be greater and the genomic DNA can move easily.

In case of PCR sample, the gene amplified won't be bulky, so, it moves fast, very fast in low percent gel, that's why to have an optimum speed we use a higher percentage of gel comparatively.

And, when our aim is to separate two sequences with relatively equal size, we must use higher percentage gel, this increases the run time, but, still we can effectively resolve two sequences with more or less equal size, say, 500bp and 550 bp.

Clear now?

Okay, how to prepare the agarose gel? It's very simple, just weigh 0.8gm of agarose and dissolve in 100 ml of TBE buffer. Microwave this until the agarose gets dissolved and you get a clear solution.
To this clear solution, add EtBr (Ethidium Bromide) 3micro liter for 25 ml (Note: while adding EtBr, the solution should not be very hot, it must not be very cool- if it gets so cool, it'll solidify, the solution must be of bearable warmth). Now, after adding EtBr, pour the gel in the gel cast with comb and allow it to solidify.

After it solidifies, add TBE buffer over it (for lubrication) and remove the comb carefully. Lanes will be formed.

Place the gel in the Agarose gel setup with anode and cathode. Load your sample DNA* in the lane.

 Load a standard DNA ladder (for finding the molecular weight of your sample DNA.)  And run at 75 V. Stop when the sample reached 3/4 th of the gel.

Now, view the gel under UV platform, the DNA band will fluoresce due to the presence of EtBr in the gel. Measure the distance travelled by your sample from the lane in cms. Also measure the distance travelled by the standard ladder.

The standard’s molecular weight is known already. So, plot a graph with Molecular weight of the standard in X axis and Rf value (Distance measured in cms) in y axis.

From the standard graph, by plotting the rf value of our sample, its molecular weight can be found.

*Preparation of DNA sample:
Take 5 micro liter of sample in an eppendorf add 5micro liter of tracking dye to it. Give a brief spin in centrifuge for mixing them.

Tracking dye contains Glycerol- which gives density to the sample, so that it goes down into the lane easily while loading; it also contains bromophenol blue which acts as a visualising agent while you are running the gel.

Got it now, any doubts? feel free to comment, I'l try to answer as soon as possible.