Refer to the diagrams for the design of a primer pair: 1) What primers could be
ID: 172136 • Letter: R
Question
Refer to the diagrams for the design of a primer pair:
1) What primers could be used to amplify all of the DNA from Lysine Codon to the STOP Codon. With the primers annotate the 5' and 3' of the primers and explain why this primer pair is optimal (e.g Tm, GC content, Self-complementarity, Primer Length).
2) Show where (to the exact base number) where each of the primers are (Start and Stop) and why they stop there. Show if they are COMPLEMENTARY or the SAME sequence as shown. Put an arrow to show which direction they are acting on and where the DNA polyermase will bind to. Label this end 5' or 3' based on how the primer pair has been designed.
pGEX-6P-2 plasmid this is a plasmid of the same family as the pGEX-2T used in the practical. A map of the plasmid and description of some features are shown on the last page. pGEX-6P-2-insert plasmid this is the pGEX-6P-2 plasmid with a 250 bp fragment inserted between the EcoRI and Xhol sites. Forward Primer-this is a primer that has been previously designed for PCR. Its location is shown on the plasmid map Reverse Primer this is a primer that has been previously designed for PCR. Its location is shown on the plasmid map Gene Ruler1 kbp DNA Ladder this is the DNA standard marker. It is shown on page 4. More details are here https www.thermofisher.com/order/catalo roduct/SM0311. The pGEX-6P-2 MCS and surrounding sequence is shown on page 4Explanation / Answer
5’-GGT GGC GAC CAT CCT CCA AAA TCG GAT CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA CTG ACT GAC GAT CTG CCT CGC GCG TTT CGG TGA TGA CGG TGA AAA CCT CTG ACA CAT GCA G-3’
Complimentary sequence of given sequence will be as given below:-
3’-CCACCGCTGGTAGGAGGTTTTAGCCTAGACCTTCAAGACAAGGTCCCCGGGGACCCTAGGGGTCCTTAAGGGCCCAGCTGAGCTCGCCGGCGTAGCACTGACTGACTGCTAGACGGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTC-5’
Plasmid used as vector:- pGEX-6P / pGEX-2T
Primer Designing from Lysine to Stop Codon
Stop Codons TAA,TAG,TGA
Lysine Codons AAA, AAG
Start Codon ATG
To design the primer we have to use one sequence from 5’-3’ end. As per requirement . the primer will be designed for sequence from Lysine to stop codon.
The primer should have cloning site sequences at the 5’ end of sequence. Those sites will be selected as per Plasmid vector(pGEX-6P or pGEX-2T).This vector have multiple cloning sites for example BamH1, ECOR1, and Xho1 sites. Suppose we have used the BamH1 so it will have particular sequence(G^GATCC).Start codon will be added also in the Sequence.
So the final Primer will as below
5’-G^GATCCATGAAATCGGATCTGGAAGTTCTG Forward Primer
5’-(MCQ Site)TCACGATGCGGCCGCTCGAGT-3’
Forward primer length 30bp
Reverse Primer 30 bp
GC content 47% Tm 70°C