Which one is the correct answer?

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ahqprion
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Which one is the correct answer?

Post by ahqprion » Wed May 23, 2007 6:33 am

This question came up from my class test.
I am not still convinced enough... so i would like to know the answer and explanation for the problem below.

Problem:
Biologist compared a gene found in the E. coli in the bean with a similar found in the E. Coli from the mouse . The first step in doing this is to do PCR on the gene.
The sequence of the gene is shown below.
5' TAGCTAGCTG GATCGATTGG TCGAATGGCTG GCGGATGCTA GGATCGATCG GCTAGAGCT 3'
3' ATCGATCGAC CTAGCTAACC AGCTTACCGAC CGCCTACGAT CCTAGCTAGC CGATCTCGA 5'

You must first design a primer for the section of the DNA you are interested in (the bolded section above). What 2 primer sequence you synthesize?
a. 3'-CTAGC-5' & 3'-ATCGT-5'
b. 5'-CTAGC-3' & 5'-ATCGT-3'
c. 5'-TAGCA-3' & 5' -GATCG -3" (i copied this exactly... double apostrophes)
d. None of the above

The teacher said the answer is C but i don't get it because the 5'-GATCG-3" is found for the last sequence which is not in the bolded section and also the sequence is reverse...
My current answer is D.
I would like to know whether this is correct answer or not.
If not, then can you give me explanation so I can prove it to teacher reasonably.
Thank you.

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Post by Dustfinger » Wed May 23, 2007 9:36 am

I'd say B is correct...
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Post by blcr11 » Wed May 23, 2007 10:26 am

I agree with the teacher. The primers in C will amplify only the sequence in bold. Remember that the 5' to 3' direction on the bottom strand reads from right to left, not left to right like it does for the top strand. So the first primer (5'-TAGCA-3' ) is the bottom strand (I would call it the 3' or reverse primer) reading from right to left and the second (5' -GATCG -3" ) is the top strand (5' or forward primer) reading right to left. I presume the double quote was a typo on the teacher's part.

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Post by blcr11 » Wed May 23, 2007 10:34 am

You would have a point about non-uniqueness if the GATCG sequence were in the reverse order, as I think you were pointing out. But a 5'-GATCG-3' primer is not the same as a 3'-GATCG-5' sequence.

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Post by blcr11 » Wed May 23, 2007 10:39 am

Grrr. Sorry. That should be that the the 5'-GATCG-3' primer is the top strand reading left to right. Haven't had the morning coffee yet.

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Post by blcr11 » Wed May 23, 2007 11:43 am

I should add--just in case it's not obvious--this has to be considered a case for learning the principles. If you really were interested in just the sequence in bold, for whatever reason, it's so short you would just synthesize the sequence directely. If you were after a larger sequence, you almost certainly would want to use primers that were longer than 5 bases (you need Tms of 55 C or greater--ususally higher that 65 C--which a 5-base sequence cannot give you). But as a teaching exercise to show the principles of pcr and primer design, this is probably OK. At least it spares the student of having to check the sequence of a 25-mer, which I can attest to being an error-prone process especially if done by eye.

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so is the c answer then ?

Post by ahqprion » Wed May 23, 2007 10:35 pm

so is the c answer then?

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Post by blcr11 » Wed May 23, 2007 11:30 pm

You mean I didn't convince you? My money is on C, like the teacher claims.

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Post by sdekivit » Sun May 27, 2007 8:11 am

the answer is indeed C, because this primer set binds both the upperstrand (1 primer is complentary to the 5' side of the upper bold strand) and the lower strand (1 primer is complementary to the 3'side of the lower bold sequence)

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Post by wbla3335 » Sun May 27, 2007 9:42 am

May I suggest a different interpretation? The question begins by saying that someone is interested in DNA sequence from 2 different sources, implying that they will be interested in differences between them. The sequence of interest is in bold. If I wanted the most information concerning possible differences between sources of DNA, I would design my primers outside the bold area, so that the data I get includes information from the entire bold area. If the primers are within the bold area, some of the data will be invariant because it will be supplied by the primers. So I would choose d and disagree with the teacher. But of course, if the teacher made up the question and says the answer is c, then the answer is c. I would just question the rationale of their choice of primers.

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Post by kanak33 » Sun Jun 10, 2007 11:19 pm

blcr11 wrote:I agree with the teacher. The primers in C will amplify only the sequence in bold. Remember that the 5' to 3' direction on the bottom strand reads from right to left, not left to right like it does for the top strand. So the first primer (5'-TAGCA-3' ) is the bottom strand (I would call it the 3' or reverse primer) reading from right to left and the second (5' -GATCG -3" ) is the top strand (5' or forward primer) reading right to left. I presume the double quote was a typo on the teacher's part.


Why does that primer 5'-TAGCA-3' would be for the bottom strand? Its exactly the same!!! What about the complementarity?
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Post by blcr11 » Mon Jun 11, 2007 9:54 am

Not sure why you think being "the same" on the bottom strand is a problem while being "the same" on the top strand is not, for can't you say "the same" thing about the other primer of the pair? Anyway, that's the way the amplification works. The first primer in c) is complemtary to the upper strand and will extend (5'-->3') making a copy of the bottom strand. The second primer is complementary to the bottom strand and makes a copy of the top when it is likewise extended. After a few cycles of pcr, most primers will bind to a pcr product that only started at one or the other end and so you end up amplifying (for the most part) only the sequence nested between the two primers. I'll grant you that trying to use 4 or 5 base primers probably isn't a very good design strategy, but I took this to be a teaching excercise.

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