Design 3 PCR primers that could be used to amplify this region of DNA. Two diffe
ID: 151225 • Letter: D
Question
Design 3 PCR primers that could be used to amplify this region of DNA. Two different primers are needed for the downstream region, so that you can preferentially amplify one sequence in the presence of the other. One "universal" prime can be designed for the upstream region. Be sure to name the primers in a way that I can distinguish them and write the sequences in the 5-3 direction. -Discuss your reason for designing the two downstream primes in the way you did. -Calculate the Tm for each of the primers. -Do you feel they meet the criteria for good primers? 5'-CGATGGTCCAGGTCGACGGTACGGTAGCT-100BP-GGTACCTGTATTGCGGTTGTTTGATTCGAA-3 5'-CGATGGTCCAGGTCGACGGTACGGTAGCT-100BP-GGTACGTATATTGCGGTTGATAGATTCGAA-3Explanation / Answer
1) one universal primer is 5'-GCTACCAGGTCCAGCTGCCATGCCATCGA-3'
FIRST REVERSE PRIMER : 5'-TTCGAATCAAACAACCGCAATACAGGTACC-3'
SECOND REVERSE PRIMER : 5'-TTCGAATCTATCAACCGCAATATACGTACC-3'
we designed our universal primer which is forward in 5' to 3' direction and it is just complementary to the given DNA sequences. the forward primer gives us 5'-3' sequence of the sense strand ( DNA strand from with protein to be made) whereas we have designed our downstream reverse primers in a reverse complementary way. The sequence got from reverse sequence is from 5'- to 3' of the complementary strand. If you just reverse it, then it will be in 3' to 5'. When you reverse complement the reverse sequence then it will be 5'-3' of the sense strand. Now both the sequences represented in 5' to 3' prime direction of the sense strand. When we translate the sequence we will get a complete amino acid sequence.
2) Tm= 2 deg(number of A and T in the primer)+4(number of G and c in the primer)
universal primer Tm=94 deg
first reverse primer Tm= 84 deg
second reverse primer Tm=86
3) no they don't meet the criteria of good primers because they have very high Tm. Ideally, Tm should fall between 50-65 deg range. above 65 degrees they have the tendency for secondary annealing.