Question

PCR Questions

What gives DNA strands its polarity? To answer, tell me what the “ 5’ ” and “ 3’ ” ends are referring to in chemical terms.  

Diagram a “template” and the primers for a PCR reaction, including the 5’ and 3’ notations as appropriate.

Will the “forward” primer have the same sequence as the coding strand or complement strand?

Will the “reverse” primer have the same sequence as the coding strand or complement strand?

After running PCR, a researcher loaded an aliquot of the reaction onto an agarose gel. In addition to the expected product, several other larger DNA bands were detected after staining. What is the most reasonable explanation for this result?

(A) Either the forward or reverse primer does not bind to the template DNA.

(B) Either the forward or reverse primer formed a double stranded DNA molecule.

(C) The annealing temperature was too low.

(D) Student forgot to add Mg²⁺ ions.

(E) None of the above is a reasonable explanation

What is EDTA, how does it work, and why is it useful for storing nucleic acids?

Answer 1

The 5' and 3' mean "five prime" and "three prime", which indicate the carbon numbers in the DNA's sugar backbone. The 5' carbon has a phosphate group attached to it and the 3' carbon a hydroxyl (-OH) group. This asymmetry gives a DNA strand a "direction". For example, DNA polymerase works in a 5' -> 3' direction, that is, it adds nucleotides to the 3' end of the molecule (the -OH group is not shown in diagram), thus advancing to that direction (downwards).