Question 1. You are sequencing a 350bp lizard gene using the Sanger dideoxy sequ

Question

Question 1. You are sequencing a 350bp lizard gene using the Sanger dideoxy sequencing method. You have amplified the region using the primer 5'-ATCCGGGCG-3'. What will be the length of the lowest band in your sequencing gel?

(A) Cannot answer the question with the provided information

(B) 350

(C) 10

(D) 9

(E) 351

Question 2. True or False

If cell has a mutated gyrase, it means that it will be unable to properly join together okazaki fragments and therefore will result in many fragmented pieces of DNA.

Question 3. Which of the following answer(s) are true (select all that apply)?

- PCR results in exclusively the desired region to be amplified.

- PCR primers must be outside the region of interest.

- PCR primers must be flanking the region of interest.

- Sequencing primers must be flanking the region of interest.

- All of the nucleotides used in Sanger Sequencing would also be able to be used for a PCR reaction where you are trying to amplify a gene of interest.

- Sanger sequencing requires dNTPs.

- A standard PCR reaction requires ddNTPs

- Small DNA fragments on a gel would be closer to the wells on a gel than larger DNA fragments.

Explanation / Answer

1.In sanger`s dideoxy method all the reactions start from the same nucleotide and end with a specific base. Thus in a solution where the same chain of DNA is being synthesized over and over again, the new chain will terminate at all positions where the nucleotide has the potential to be added because of the integration of the dideoxynucleotides. Dideoxy nucleotides lack a hydroxyl group on the 3’ carbon of the sugar ring thus when these are added to the growing chain no further nucleotide can be added. Thus these are chain terminating and terminates the chain where dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. Finally, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA. The band of shortest fragments are at the bottom of the autoradiogram

Here the length of the lowest band in your sequencing gel would be 9 on the basis of information given above.

2. False

Gyrases belong to the class topoisomerases. Their main function is to catalyze the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Okazaki fragments are joined together by ligase enzyme so if there is a mutated gyrase there will be no effect on joining of Okazaki fragments. These will join properly.

.3.True statements are-

PCR results in exclusively the desired region to be amplified.

PCR primers must be flanking the region of interest.

false statements are-

Sanger sequencing requires dNTPs.-This requires ddNTPS.

A standard PCR reaction requires ddNTPs- this requires dNTPs..

Small DNA fragments on a gel would be closer to the wells on a gel than larger DNA fragments.-larger DNA fragments would be closer to the wells on a gel as these will move slower than the smaller fragments.

All of the nucleotides used in Sanger Sequencing would also be able to be used for a PCR reaction where you are trying to amplify a gene of interest.- few sequences are used .

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