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# Reverse transcriptase polymerase chain reaction (RT-PCR) of a UV-dependent gene

## Problem

A graduate student interested in skin cancer and melanin synthesis used reverse transcriptase polymerase chain reaction (RT-PCR) to measure how UV light exposure stimulated production of melanin. Specifically, she wanted to determine the effect of UV light on the transcription of the gene for pro-opiomelanocortin (POMC) in keratinocytes. As a post-translational modification, POMC is cleaved to yield multiple active peptides, including melanocyte-stimulating hormone (α-MSH), which can then trigger synthesis of melanin in nearby melanocytes.
Figure 1 Schematic of RT-PCR experiment. The position of the reverse transcriptase primer (Primer 1) and the positions of the PCR primers (Primers A and B) are shown.
The student exposed keratinocytes to ultraviolet light of a constant intensity for various lengths of time. After this UV exposure, she harvested cells and conducted RT-PCR, using primers specific for a fragment of POMC spanning an intron (Figure 1). She then analyzed her sample by gel electrophoresis, as shown in Figure 2. She included a DNA ladder of eight DNA fragments at 100 base pair intervals, ranging from 100 to 800 base pairs. She also included positive and negative controls, in lane 6 and lane 7 respectively.
Figure 2 Results of gel electrophoresis
Assuming all are approximately 8 nucleotides in length, which of the following would be effective as primers during the reverse transcription step of the experiment described above?
I. A multitude of random, scrambled primers
II. Primers specific to the first intron of POMC
III. A string of thymine nucleotides
IV. The same set of primers as used in the PCR amplification step