which of the three primers would you choose? give a reason for your choice and a
ID: 58084 • Letter: W
Question
which of the three primers would you choose? give a reason for your choice and also give a reason why the other two are unsuitable?
2) what sort of the result would you expect if extension step shortened from 72C 5 minutes to 3 minutes?
You are planning another site-directed mutagenesis experiment and you are supplied with three sets of primer pairs. The sequences of one primer of each pair are as indicated below 1: 5'-AACTGGATACACTAGCGACATGCcCAGAGACATAACTCACTGAGG- 2: 5 ,-GAATCTACACACTCGGGGGGGGATCCCCCCCCCCCTAAATCTACC-3 , 3: 5'-ACTAGCCTCAATAGTCAGACTACAACATAGATACAGCATATAATA-3 3'Explanation / Answer
1. I will use the first primer pair, beacuse it has right amount of GC content (49%), its melting temperature is about 70oC and it forms very less self complementary structures like primer dimers.
Other two primers are not suitabole beacuse, the second primer has a very high GC content of 62% and also it forms many potential primer dimers beacuse of repeatative GC sequences, the third primer is not suitable beacuse of very low GC content of 33%.
2. If the extension step is shortened from 5 mins to 3 mins at 72oC, the PCR amplication will not be correct and the amplicons will be shorter in length. As the primer is of 45 bps, so it requires more time to extend the sequence during the extension step.
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