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20.109(F08): Mod 1 Day 1 DNA engineering using PCR: Difference between revisions
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20.109(F08): Mod 1 Day 1 DNA engineering using PCR (view source)
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In addition to the target, PCR requires only three components: primers to bind sequences flanking the target, dNTPs to polymerize, and a heat stable polymerase to carry out the synthesis reaction over and over and over. PCR is a three-step process (denature, anneal, extend) and these steps are repeated 20 or more times. After 30 cycles of PCR, there could be as many as a billion copies of the original target sequence. | In addition to the target, PCR requires only three components: primers to bind sequences flanking the target, dNTPs to polymerize, and a heat stable polymerase to carry out the synthesis reaction over and over and over. PCR is a three-step process (denature, anneal, extend) and these steps are repeated 20 or more times. After 30 cycles of PCR, there could be as many as a billion copies of the original target sequence. | ||
[[Image:PCRcycle.png|thumb| | [[Image:PCRcycle.png|thumb|right|500px| '''PCR cycle''']] | ||
Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is only 20 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight. | Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is only 20 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight. | ||
[[Image:Be109karymullis.jpg|thumb| | [[Image:Be109karymullis.jpg|thumb|left|150px|'''Kary Mullis''']] | ||
''"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." '' --Kary Mullis from his Nobel lecture; December 8, 1983 | ''"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." '' --Kary Mullis from his Nobel lecture; December 8, 1983 | ||
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#Sketch the expected product from the PCR you performed, clearly indicating the 5’ and 3’ end. Include the restriction sites that you have introduced and the expected length of the product. | #Sketch the expected product from the PCR you performed, clearly indicating the 5’ and 3’ end. Include the restriction sites that you have introduced and the expected length of the product. | ||
#Read NEB's [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/setting_up_reaction.asp "Setting up a Restriction Endonuclease Reaction"] | #Read NEB's [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/setting_up_reaction.asp "Setting up a Restriction Endonuclease Reaction."] | ||
#You will write up the work you do in Module 1 as a brief lab report and a powerpoint pitch. To help you pace your work, as well as give you feedback early on, you will be required to draft small portions of the report as homework assignments. For this time, you should write the sub-section of your Materials and Methods (see #6 under Order of Assembly [[20.109(F08):DNA_engineering/Lab_report | here]] and general guidelines [[20.109(F08):Guidelines for writing a lab report#Materials_and_methods |here]]) that describes the PCR you did. This will require explaining the basic design elements for your primers, in a sufficiently informative way such that a classmate would have the necessary information to make their own primers that serve the same purpose (even if the primers are not identical). | #You will write up the work you do in Module 1 as a brief lab report and a powerpoint pitch. To help you pace your work, as well as give you feedback early on, you will be required to draft small portions of the report as homework assignments. For this time, you should write the sub-section of your Materials and Methods (see #6 under Order of Assembly [[20.109(F08):DNA_engineering/Lab_report | here]] and general guidelines [[20.109(F08):Guidelines for writing a lab report#Materials_and_methods |here]]) that describes the PCR you did. This will require explaining the basic design elements for your primers, in a sufficiently informative way such that a classmate would have the necessary information to make their own primers that serve the same purpose (even if the primers are not identical). | ||
