4. You perform a PCR reaction using the following template DNA and primers. What
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Question
4. You perform a PCR reaction using the following template DNA and primers. What size(s) of products do you expect? Explain vour answer. Primer: 5'-ctctgggctc agagcagccc-3 Primer: 5'-catgcagccg tcctgatctc-3 Template: 1 attatatttt atattatata ctctgggctc agagcagccc 40 41 atattatata tatatatttt aaaatattat aaatttattt 80 81 catgcagccg tcctgatctc attatatttt ataatttttt 120 121 ttttattttt attatatttt ttttttattt ttattttttt 160 161 aaaatattat tatattatta taattaattt attataaaat 200 201 tatatatttt attatatttt tatatatttt attaaatttt 240 241 attatatttt tttttaattt ttattattat tttttatata 280 281 tatatatttt tatatatttt attatatttt attatatttt 320 321 tttatataaa attatatttt tatttttttt ttttttataa 360 361 gggctgctct gagcccagag attatatttt attataaaat 400 401 attatatttt attataaaat aaaatataat aaaatattat 440 441 aaaatattat aaaaaaaaat gagatcagga cggctgcatg 480 481 atattatata atattatata attatatttt atattatata 500Explanation / Answer
Polymerase chain reaction (PCR) used to amplify the DNA in a cell-free system and it is an alternate method for gene cloning. It was first discovered by the Kary Mullis in 1983 and he awarded with the noble prize in 1993. The requirements for polymerase chain reaction are genomic DNA, a forward primer and reverse primer, PCR buffer, dNTPs, Taq DNA polymerase, deionized water and a thermal cycler.
Template Strand (Genomic DNA)
ATTATATTTTATATTATATACTCTGGGCTCAGAGCAGCCCATATTATATATATATATTTTAAAATATTATAAATTTATTTCATGCAGCCGTCCTGATCTCATTATATTTTATAATTTTTTTTTTATTTTTATTATATTTTTTTTTTATTTTTATTTTTTTAAAATATTATTATATTATTATAATTAATTTATTATAAAATTATATATTTTATTATATTTTTATATATTTTATTAAATTTTATTATATTTTTTTTTAATTTTTATTATTATTTTTTATATATATATATTTTTATATATTTTATTATATTTTATTATATTTTTTTATATAAAATTATATTTTTATTTTTTTTTTTTTTATAAGGGCTGCTCTGAGCCCAGAGATTATATTTTATTATAAAATATTATATTTTATTATAAAATAAAATATAATAAAATATTATAAAATATTATAAAAAAAAATGAGATCAGGACGGCTGCATGATATTATATAATATTATATAATTATATTTTATATTATATA
Forward Strand 5’-CTCTGGGCTCAGAGCAGCCC-3’
Reverse Strand 5’-CATGCAGCCGTCCTGATCTC-3’
The primer binding sites on DNA in above template is represented by the italics and the amplified region in bold letters. The PCR product size is 460 base pairs.
To the reaction mixture, an appropriate amount of the given template DNA is added and the reaction containing PCR tubes are placed in PCR machine. Once the PCR starts initially the DNA is subjected to the 94C for 5 minutes which cause the unwinding of DNA template. Once the DNA is unwound immediately the forward and reverse primers bind (italics) to the appropriate sites on template DNA. The Taq DNA polymerase extends the 3’ hydroxyl groups of primers which bound to template DNA. After the successive rounds of PCR cycles, the product size is 460 base pairs.
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