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Explanation of the procedure behind PCR includes discovery, definition, requirements , steps and applications
http://www.biologyexams4u.com/2014/04/pcr-polymerase-chain-reaction.html
Multiple choice questions on PCR (Polymerase Chain reaction)
http://www.mcqbiology.com/2013/01/mcq-on-pcr-polymerase-chain-reaction.html
Biotechnology Notes:
http://www.biologyexams4u.com/2011/09/biotechnology-notes.html
Biotechnology Multiple Choice Questions:
http://www.mcqbiology.com/2012/11/mcq-on-biotechnology.html
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0:00
Today the topic of our discussion is polymerized chain reaction and the theory behind this reaction
0:05
Polymerized chain reaction was developed by caramulus in 1983. It is a DNA, in vitro DNA amplification
0:11
technique that allows to synthesize millions of copies of a gene or DNA from a single copy. Previously
0:17
the technique was gene cloning that approximately take more than a week to synthesize multiple copies of a gene
0:24
This is a revolutionary technique by all means as this technique takes less than two hours to
0:29
to synthesize millions of copies of a particular gene. This is called as a polymerized reaction because only enzyme used in this reaction is DNA polymerized
0:40
Now let us look into the reaction components or the requirements for PCR
0:45
First of all, the target DNA that should contain the sequence to be amplified
0:49
Two oligonucleotide primers to initiate the DNA synthesis as no DNA polymerize is capable of initiating a DNA synthesis
0:57
DnTPs which are the building blocks of nucleotides or DNA. Then a thermostable DNA polymerize
1:05
this reaction takes place at approximately 92 degrees Celsius. Normal enzymes could not withstand this temperature
1:12
and this enzyme tag polymerase which was isolated from a thermophilic bacterium
1:16
called thermosacquarticus and that could withstand 94 degrees Celsius Then magnesium ions this is a co of the enzyme There are many enzymes like Wend PfU et cetera instead of TAC polymerase
1:31
And buffer solution, and this actually maintains the pH of the solution
1:36
For a DNA synthesis to occur, there should be a primer. And this is a requirement
1:41
No non-DNA polymerize is capable of initiating a DNA synthesis. There should be a short stretch of DNA which, with
1:49
free 3-o-h-end so that further nucleotides can be added by a DNA polymerize and this is a synthesis
1:56
and the primer determines a part of the DNA molecule that is to be copied so we should know a short stretch of
2:02
sequence of the target to be amplified so that we could synthesize the primer accordingly
2:09
and this is a PCR tube and we are actually mixing all the components in this tube and just keep that
2:15
in this thermocycler PCR machine which is called as a thermocir adjust the settings it is as simple as that and this is the reaction the procedure
2:27
behind PCR and this is our DNA strand and I have given this 5-3-dash prime and that is
2:34
very important the first step is we are actually heating up the reaction to 94
2:40
degrees Celsius for one minute and this will denature the strands that is by the breakage of hydrogen bonds second step is primer annealing cool down the solution to 56 degrees Celsius
2:53
that will initiate the binding of primer. We have synthesized the primer in accordance with this sequence
3:00
and primer will bind to the sequence at a 3-dash end so that there will be a free 3-o-H end for the DNA polymerase activity
3:08
And the third step is the elongation, increase the temperature to 72 degrees
3:13
Celsius, TAC polymerase, DDAG DNA polymerase is optimum temperature and we have added all the dnTPs so that the synthesis will occur in 5.3-dars direction
3:24
And this constitutes a single PCR cycle. And this is the steps explained in detail
3:32
First step that is a denaturation heating up to 94 degrees Celsius for a minute
3:38
And this will denatures double-stranded DNA into single strand by breakage of hydrogen bonds
3:42
The second step is primer annealing where the primer binds to the 3-end of the target DNA molecule
3:50
Third step is elongation by DNA polymerize, which could withstand a temperature of 72 degrees Celsius
3:59
and the further DNA molecules are nucleotides are added by DNA polymerize
4:04
And duration of a single cycle is approximately 4 to 5 minutes and the time for each step may vary depending on the primer gc content of the primer and many other aspects And this constitutes a single step
4:17
And the DNA amplification will take place exponentially to 816 like that
4:24
And reaction usually consists of 30 to 35 cycles, and we'll be getting millions of copies of our chain of interest
4:31
And these are the applications most molecular biology lab. This is a common procedure, day-to-day procedure
4:37
and new applications are created every day and there are many variations of PCR nowadays
4:43
And to amplify DNA fragments isolated from organisms, maybe for gene manipulation techniques
4:49
for the determination of, sex determination of embryo, in forensic science that is in DNA profiling or DNA fingerprinting
4:55
and in mapping genes, in eustace probes and many, many other other applications
5:03
And this is what happens in a PCR cycle. First cycle, the number, the number of the number of, of DNA molecule newly formed is two and the second cycle it will be four, eight
5:12
16 and this the number of copies will grow exponentially and at the end of 30 cycles
5:18
we'll be getting millions of copies of our particular gene that can be further screened
5:22
using electrophoress and can be isolated and can be used for further research and this is
5:28
the graph. The only limit is the PCR reagents. Hope things are clear you are with biology exams
5:34
sorry.com thank you so much for watching
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