You are studying a new gene \"X\" that you think controls skin color in Bearded

Question

You are studying a new gene "X" that you think controls skin color in Bearded es you need lots of gene X DNA to work with. So you Dragons. In order to determine what gene X do decide to amplity it through PCR You have a forward and a reverse primer that have been designed to gene Xf- a similar gene in frogs. You are pretty sure that these two primers will work in Bearded Dragons, so you decide to go ahead with the PCR. In frogs, the Forward Xf primer binds gene Xf between nucleotides 32 and 53. The Reverse Xf primer binds between nucleotides 642 d 663. When you analyze the PCR reaction you set up using Bearded Dragon DNA, your PCR product is 700 bp. Based on this information, answer the following: a. What size PCR product would these two primers create if they were used to amplify the frog gene Xf?? Forward primer NT. Reverse primer NT: PCR Product Size: bp b. Based on this predicted frog PCR product and the Bearded Dragon PCR product you created, do you think that these two primers amplified gene X in the Bearded Dragon? Why or why not? c. A fellow lab worker brings you DNA containing what might be a similar gene in Leopard Geckos (Xa). She asks you to see if you can amplify it using the same primers you used in frogs and the Bearded Dragon. You run the PCR and then analyze the product by running the DNA on an agarose gel. As a control, you run out the PCR product you amplified in Bearded Dragons and a DNA "ladder" of DNA pieces of known size. The gel results are shown below. Marker DNA ladder; Lane A Bearded Dragon PCR product; Lane B Leopard Gecko PCR results. In the box below, determine the sizes of the PCR products shown in Lanes A & B. sampies Marker A Size of Bearded Dragon DNA: Sizes of Leopard Gecko DNA: Do you think your PCR of Leopard Gecko gene X worked? Why or why not?

Explanation / Answer

7)a) Ans: Information about the frog primers are -

Forward primer(xf-F): binds between 32-53 bp

Reverse primer(xf-R): binds between 642-663 bp

So there is no specific mention of the primers exact binding location. Now based on this data we can assume 3 cases: 1) forward primer binds to position 32bp & reverse in 642 bp; so resulting amplified product= (642-32)=610 bp; Likewise, forward primer binds to position 53bp & reverse in 663 bp; so resulting amplified product= (663-53)=610 bp;

2) forward primer binds to position 32bp & reverse in 663 bp; so resulting amplified product= (663-32)=631 bp;

3) forward primer binds to position 53bp & reverse in 642 bp; so resulting amplified product= (642-53)=589 bp;

So the size of amplified PCR product would be in a range of 589bp to 631 bp.

b) Ans: Well from our first assumption it is clear that the amplified product in frog is around 589-631 bp, and it is given that the size of the amplified bearded dragon is 700 bp. So it is an indication that the primers we used did work but we can't be sure whether the amplifies band is a compact bearded dragon gene, as no specific binding position is mentioned in frog primers, so it could bind to any suitable complementary region in the bearded dragon DNA. Also in the question, it is not mentioned how much similarity lies between the frog DNA and the bearded dragon DNA. So it is most likely that those primers will not be able to generate the exact X gene of the bearded dragon.

C) Ans: From the agarose gel picture it is clear :

The size of  bearded dragon gene: 700 bp

The sizes of amplified leopard GECKO genes are =800 bp, slightly more than 400bp, slightly more than 200bp.  

So it is sure that the primers used to amplify the leopard GECKO gene didn't work, otherwise, it would have yielded a single distinct band. Presence of multiple bands indicates that the primers bind to the leopard DNA in more than one place according to its complementarity with it.

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