RT-PCR analysis of POMC cDNA
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- RT-PCR analysis of POMC cDNA
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RT-PCR analysis of POMC cDNA
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[post_date] => 2024-12-23 09:09:11
[post_date_gmt] => 2024-12-23 14:09:11
[post_content] => Practice Passage (Question 1-6)
*This passage is the property of Khan Academy and has been reformatted into an AAMC-style interface in their entirety by MedLife Mastery. MedLife Mastery does not endorse and is not an affiliate of Khan Academy.
Researchers studying skin cancer and melanin synthesis used reverse transcriptase polymerase chain reaction (RT-PCR) to measure how UV light exposure stimulated the production of melanin. Specifically, they wanted to determine the effect of UV light on the transcription of the gene for pro-opiomelanocortin (POMC) in keratinocytes. During 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.
The researchers exposed keratinocytes to ultraviolet light of a constant intensity for various lengths of time. After this UV exposure, they harvested cells and conducted RT-PCR, using primers specific for a fragment of POMC spanning an intron, as shown in Figure 1.
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.
They then analyzed their samples by gel electrophoresis, as shown in Figure 2. They included a DNA ladder of eight DNA fragments at 100 base-pair intervals, ranging from 100 to 800 base pairs. They also included positive and negative controls, in lane 6 and lane 7, respectively.
Figure 2 Results of gel electrophoresis
[post_title] => RT-PCR analysis of POMC cDNA
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[question] => Which of the following could 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
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[answer] => 4
[description] => Reason for Correct Answer:
Reverse transcription involves making cDNA from an mRNA template. Therefore, the primer would need to bind to mRNA.
After genes are transcribed, introns are spliced out of the nascent mRNA. Therefore, primers specific to the first intron of POMC (Option II) would not target mRNA.
Besides splicing, another important post-transcriptional modification is poly-adenylation. This process results in a “poly-A tail,” or a string of adenines. For this reason, primers consisting of strings of thymine (Option III) or uracil will bind to mRNA.
Primers containing strings of thymine (T) are often referred to as poly(T) primers. Poly(T) primers are designed to anneal to the polyadenine (poly(A)) tail found at the 3′ end of most eukaryotic mRNA molecules.
Option IV uses primers that are specific for the each strand of cDNA, which will also bind mRNA and therefore produce the desired RT-PCR product. (The mRNA sequence will correspond to one of these cDNA sequences.) Note that Option IV refers to primers A and B in Figure 1.
Option I (random primers) would also likely work, as one primer would likely bind to mRNA. PCR typically uses specific primers designed to match the sequences flanking the target DNA region that needs to be amplified. (These primers are usually 18-25 nucleotides long.) However, there are variations of PCR, where random primers are used. In these techniques, short, random sequences of oligonucleotides (usually 6-8 nucleotides long) are used as primers. This is often used in techniques like random amplification of polymorphic DNA (RAPD) analysis or cDNA synthesis during reverse transcription PCR (RT-PCR).
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[answers] => Array
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[0] => Array
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[each_answer] => A. IV only
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[each_answer] => B. II and IV only
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[2] => Array
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[each_answer] => C. I, II, and III only
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[each_answer] => D. I, III, and IV only
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[1] => Array
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[quiz_unique_key] => 3243476205
[question] => What would be the most effective negative control for the experiment described above?
[value] => Array
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[answer] => 3
[description] => Reason for Correct Answer:
In general, a negative control is designed such that no result is expected to occur because the hypothesized causal factor is taken away. Other than this, the negative control conditions should be the same as the experimental conditions.
An effective negative control addresses the experimental hypothesis. In this case the hypothesis can be inferred to be that UV exposure influences POMC expression in keratinocytes.
If the hypothesis concerns UV dependent transcription of POMC, then one must first know about UV independent transcription of POMC.
A suitable negative control would be an extract from keratinocytes that had not been treated with UV light.
)
[answers] => Array
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[0] => Array
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[each_answer] => A. A known sample of POMC mRNA
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[1] => Array
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[each_answer] => B. Extract from melanocytes post-UV exposure
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[2] => Array
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[each_answer] => C. Extract from keratinocytes that had not been treated with UV light
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[3] => Array
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[each_answer] => D. Extract from keratinocytes that had been killed with UV light
)
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[2] => Array
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[quiz_unique_key] => 2187790141
[question] => One interpretation for the negative result in lane 1 is that a 15-minute exposure time to UV light is insufficient for induction of POMC transcription. Which of the following is a necessary assumption of this interpretation?
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[answer] => 2
[description] => Reason for Correct Answer:
An assumption is necessary if it must be true in order for the explanation to be true.
In this case, the explanation is that 15-minute exposure time is not long enough for UV light to induce POMC transcription.
To make this conclusion, one must assume that there are not other factors besides length of time contributing to the lack of POMC in lane 1.
For instance, there is an assumption that the lack of POMC in lane 1 was not caused by the fact that the wavelength of light wasn’t short enough to induce POMC transcription in this time frame.
)
[answers] => Array
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[0] => Array
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[each_answer] => A. POMC transcription rate is inversely related to the wavelength of the applied light.
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[1] => Array
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[each_answer] => B. Light of a sufficiently short wavelength was used to induce POMC transcription.
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[2] => Array
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[each_answer] => C. Post-translational POMC cleavage is correctly occurring.
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[3] => Array
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[each_answer] => D. Treatment with UV light did not induce apoptosis in any of the keratinocytes.
)
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[3] => Array
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[quiz_unique_key] => 1579828684
[question] => Why might lane 3 in Figure 2 have two bands, while other lanes have one or none at all?
[value] => Array
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[answer] => 3
[description] => Reason for Correct Answer:
Gels are usually shown vertically with the assumption that the samples were added to the top of the gel. Because gel electrophoresis separates analytes based on size, we know that the bands higher up on the photo correspond to larger components.
If the sample were contaminated with RNAses, the resulting fragments would each be shorter than the desired fragment.
Primer dimers would also result in shorter sequences and would therefore be lower on the gel than the desired fragment.
Intron 1 is outside of the region amplified by the PCR primers, and would therefore not influence the size of the final, desired fragment.
The only plausible explanation for the larger band in lane 3 is that the sample was contaminated with genomic DNA, which would be larger in size than the cDNA of interest.
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[answers] => Array
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[0] => Array
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[each_answer] => A. Alternative splicing gave an mRNA that lacked intron 2, but contained intron 1.
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[1] => Array
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[each_answer] => B. The sample in lane 3 was contaminated with RNAses, which degraded the mRNA product to give two fragments.
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[2] => Array
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[each_answer] => C. The sample in lane 3 was contaminated with genomic DNA.
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[each_answer] => D. The primers hybridized to each other and were amplified, generating many “primer dimers,” along with the desired fragment.
)
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[4] => Array
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[quiz_unique_key] => 1498560436
[question] => Suppose that α-MSH is found to activate POMC transcription. With this information in mind, which is the most accurate graph of the data from Figure 2?
[value] => Array
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[answer] => 3
[description] => Reason for Correct Answer:
The brightness of the bands on the gel indicates the amount of α-MSH present at that condition. Notice that there is no α-MSH until the 30-minute mark, which matches all of the graphs.
α-MSH is a product of POMC transcription. Because α-MSH also activates POMC transcription, POMC transcription should have positive feedback on itself (i.e. POMC transcription stimulates more POMC transcription).
This matches the gel electrophoresis results, which show an exponential increase in brightness in the bands as the time of UV exposure increases.
Since α-MSH activates transcription of POMC, the amount of POMC mRNA will also increase exponentially as UV exposure increases, as shown in the graph in Choice C.
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[each_answer] => A.
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[each_answer] => B.
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[each_answer] => C.
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[5] => Array
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[quiz_unique_key] => 3044259542
[question] => Suppose the researchers used quantitative PCR to evaluate the level of cDNA in the experiment (instead of standard PCR followed by electrophoresis). Which of the following figures shows the expected outcome of this experiment?
[value] => Array
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[answer] => 2
[description] => Reason for Correct Answer:
Quantitative PCR (qPCR) is a method for detecting the levels of specific DNA molecules or sequences. Specific primers are used, along with a dye that becomes fluorescent when it binds to double-stranded DNA.
An increase in fluorescence corresponds to an increase in double-stranded DNA. This means that the target DNA sequence is present and is being amplified by the PCR reaction.
The fluorescence of samples with higher target DNA concentrations will pass the detection threshold after fewer cycles. Samples with lower concentrations of the target sequence will take longer to emit fluorescence. The number of cycles required for fluorescence to pass the threshold can be compared with standards to calculate the exact DNA concentrations of the samples.
Based on the intensities of the bands in Figure 2, the correct order of DNA concentrations is: (-)/1, 2, 3, (+), 4, 5 (ascending order).
This corresponds to the qPCR chart in which sample 5 becomes fluorescent in the fewest number of cycles.
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[each_answer] => D.
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Figure 1
Figure 2
