FL6 B/B Q18

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hannahm
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Joined: Sun Jan 05, 2020 7:25 pm

FL6 B/B Q18

Post by hannahm » Sun Apr 19, 2020 1:41 pm

Hi, I have a question on question 18 of FL6. I do not understand how the answer could be A. since reverse transcriptase is going from RNA to DNA?

Question 18


If the coding strand for a certain gene begins with 5’ AGC CTT CGG CTG ACT GGC TGG, which of the following is a possible primer that researchers could use for reverse transcription PCR amplification?

A. 5’ AGC CTT CGG CTG ACT GGC TGG 3’


B. 5’ TCG GAA GCC GAC TGA CCG ACC 3’


C. 5’ AGC CUU CGG CUG ACU GGC UGG 3’


D. 5’ CCA GCC AGU CAG CCG AAG GCU 3’
NS_Tutor_Mathias
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Re: FL6 B/B Q18

Post by NS_Tutor_Mathias » Mon Apr 20, 2020 5:34 pm

The question asks about reverse-transcription PCR amplification (RT-PCR). That means that the procedure will be using RNA and turning it into DNA before amplification. That means the complement to the given coding strand will be our RNA. That in turn means the primer for this process will be identical to the DNA coding strand except for the thymine->uracil replacement.
hannahm
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Re: FL6 B/B Q18

Post by hannahm » Mon Apr 20, 2020 10:16 pm

hmm ok I see. but how were we supposed to know from that question that we are interested in making the primer? I read that question as what RNA primer would lead to that coding strand using reverse transcriptase?
NS_Tutor_Mathias
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Re: FL6 B/B Q18

Post by NS_Tutor_Mathias » Thu Apr 23, 2020 6:17 am

I apologize for being late to continuing this discussion. I've tried to post twice and lost the message both times so far.

The key idea at work is that RT-PCR couldn't possibly be performed without an RNA template. If we used the coding strand that we're given, it would just be regular PCR, and we wouldn't actually know what primer to make, because we are only given an area upstream of the sequence. We don't know what the 5' side of the template side actually looks like.

So what you would want to understand here is that answering this requires predicting what the primary RNA transcript of this coding strand will look like, and that the primer used in RT-PCR will have to be complementary to this primary RNA transcript. Since the transcript itself is complementary to the coding strand, we are looking at the complement to the complement, also known as "exactly the same as the original". Except of course that our primer must be made of RNA, so we replace T with U.

(Bonus question: Notice that I said 'primary transcript' rather than 'mRNA'. In what way are these two things not identical?)
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