1. What are the four main reagents of the PCR 2.What is the name of the first st
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Question
1. What are the four main reagents of the PCR 2.What is the name of the first step of thermal cycling? Describe what occurs in this step. 3.What is the name of the second step of thermal cycling? Describe what occurs in this step. 4.Whay is the name of the third step of thermal cycling? Describe what occurs in this step. 5. What are some beneficial uses of PCR technology. Review 1. What are the four main reagents of the PCR? 2. What is the name of the first step of thermal cycling? Describe what occurs in this step. 3. What is the name of the second step of thermal cycling? Describe what occurs in this step. 4. What is the name of the third step of thermal cycling? Describe what occurs in this step . What are some beneficial uses of PCR technology? Why did we use Escherichia coli as our (+) control?Explanation / Answer
Ans. 1
Primers, Templeat DNA, Taq Polymerase & denucleotides tri -phospate (dNTPs) are the main components of PCR reaction mixture. but other than these four you will need PCR buffer to maintain the pH of mixture, MgCl2 as a cofactor of Taq DNA pol. and water to make a final reaction volume.
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Ans 2.
first reaction is known as a DENATURATION. in this step, temperature goes high (generally 940 to 950 C). because of high temperature, double stranded DNA will unwind and become a single stranded, so we can have a chance of primer binding.
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Ans 3.
2nd step of PCR is known as an ANNEALING, it means the binding or joining. in this step the temperature comes down (generally 550 to 600C). because of low temperature and presence of single stranded templet will give chance to primers for finding their complimentary strand in templet and bind to that perfect sequence. this step is most important and most variable for most of different PCR templets. generally the annealing temperature is depends on the Tm of primers.
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Ans 4.
Third step is known as Extension, as availability of primer bound single stranded DNA, and a free 3' end of primer gives a Taq Pol. to extend the primer end up to temperature supports. in this step, temperature goes high to 720C, which is optimum temperature for Taq Polymerase. so strands will generated and at the end of this step, you will have two hybrid (parent - daughter) DNA molecules from single parent molecule.
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Ans 5.
there are number of uses of PCR. even if you can understand the whole process then you can use the same technology by your own way.
suppose if you need to find that Tuberculosis bacteria is present or absent in yout sputum sample, then you can simply use that sputum sample for DNA extraction, and by that DNA sample you can run PCR with Tuberculosis Specific primers ( which binds to only tuberculosis DNA). now if you get amplification (a band on agarose gel) then it means your sputum sample is ifected with Tuberculosis bacteria. this is the example of benificially use of PCR in diagnosis. simmilarlly you can diagnose viral infection, fungi infection and many more....
if you are doing research, and you get an unknown bacterium, then you can extract its DNA, and by using Universal bacterial Primer, you can amplify the DNA of that unknown bacterium, which can be sequenced later to identify that perticular bacterium. thus it helps a lot in research too. you can identy a plant variety by this technology too.
so there are enourmous applications of PCR technoly now a day.
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Ans. 6
How can i know that what experiment you going to perform, beside that knowledge i can't answer this que correctly, although i am trying to judge and answer accordingly.
may be you all are going to learn PCR so you need the Templet DNA, for amplification, so you are using bacterial DNA as a positive control, because you will have a primers for Bacteria.
or may be you are going to amplify the unknown DNA, so you need to confirm that nothing is mishandled in practical, so to confirm that you put the positive control, and so you use this.
whatever it is,
but the main thing is...
positive controlused to confirm the viable activity of all reagents used in reaction, satisfactory working of thermal cycler machine, and the perfect handling.
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