Your friend is trying to amplify a DNA sequence using PCR, but he didn’t feel to
ID: 257790 • Letter: Y
Question
Your friend is trying to amplify a DNA sequence using PCR, but he didn’t feel too confident about designing the primers, so he ordered two different sets of primers (4 primers total). Unfortunately, neither set produced a visible product when ran on a gel through electrophoresis. Below are the primers that he used and the DNA to be amplified:
DNA template:
5’ ATTCGGACTTG----(750 bases to amplify)----GTCCAGCTAGAGG 3’
3’ TAAGCCTGAAC--------------------------------------CAGGTCGATCTCC 5’
Primer pair #1 (a) 5’ GGACTTG 3’ (b) 5’ GTCCAGC 3’
Primer pair #2 (a) 5’ TTCAGGC 3’ (b) 5’ AGCTGGA 3’
a) With the DNA denatured as below, show where primer pair #1 should anneal. Would this end up in a PCR product, why or why not?
5’ ATTCGGACTTG----(750 bases to amplify)----GTCCAGCTAGAGG 3’
3’ TAAGCCTGAAC--------------------------------------CAGGTCGATCTCC 5’
b) With the DNA denatured as below, show where primer pair #2 should anneal. Would this end up in a PCR product, why or why not?
5’ ATTCGGACTTG----(750 bases to amplify)----GTCCAGCTAGAGG 3’
3’ TAAGCCTGAAC--------------------------------------CAGGTCGATCTCC 5’
Explanation / Answer
5’ ATTCGGACTTG----(750 bases to amplify)----GTCCAGCTAGAGG 3’
3’ TAAGCCTGAAC--------------------------------------CAGGTCGATCTCC 5’
5’ GGACTTG 3’ 5’ GTCCAGC 3’
This primer set would not amplify the double stranded DNA because both the primers are bound to the same strand. Each primer should bind to each strand so that both the strands would get amplified.
b.
5’ ATTCGGACTTG----(750 bases to amplify)----GTCCAGCTAGAGG 3’
3’ AGGTCGA 5’
3’ TAAGCCTGAAC--------------------------------------CAGGTCGATCTCC 5’
5’ TTCAGGC 3’ would bind to 3’ AAGTCCG 5’. However, this sequence is not found in the given DNA sequence. So this primer would not bind anywhere. Therefore, it cannot generate an amplified PCR product.
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