Can you please help me with these questions and explain how to read a gel electr
ID: 311179 • Letter: C
Question
Can you please help me with these questions and explain how to read a gel electrophoresis pattern and restriction maps, thank you
5. The agarose gel at the right shows the pattern of bands obtained when a particular fragment of double- stranded DNA is digested to completion with the restriction enzymes shown a. Which of the six restriction maps shown below is consistent with the observed pattern of restriction fragments? 10 12 kb LT 1 10 12 kb A N 10 12 kb 0.5 N A E-ELOR 10 12 kb A N 10 2 kb E A N N A E 8 kbExplanation / Answer
Explanation of how to read a gel electrophoresis pattern and restriction maps:
We have 6 restriction maps:
1. restriction map: (of 12 kb fragment size)
If we use E restriction enzyme: We can cleave the given DNA into 3 fragments one is 6 KB and other is 5 kb and the other is 1 kb. So it cleaves at 6 kb and 11 kb site
If we use A restriction enzyme: We can cleave the given DNA into 2 fragments one is 8 KB and other is 4 kb so it cleaves at 8 kb site
If we use N restriction enzyme: We can cleave the given DNA into 2 fragments one is 3 KB and other is 9 kb. so it cleaves at 3 kb site
Similarly do for all, presenting simply in table format
Restriction map in order from top to bottom
Length Kb
E restriction enzyme
A restriction enzyme
N restriction enzyme
Cleaves at
Fragments size
Cleaves at
Fragments size
Cleaves at
Fragments size
1
12
6kb
11 kb
6 kb
5kb
1 kb
8kb
8 kb
4 kb
3 kb
3 kb
9 kb
2
12
6 kb
11 kb
6 kb
5 kb
1 kb
3 kb
3 kb
9 kb
8 kb
8 kb
4 kb
3
12
6 kb
11 kb
6 kb
5 kb
1 kb
8kb
8 kb
4 kb
9 kb
9 kb
3 Kb
4
12
6 kb
11 kb
6 kb
5 kb
1 kb
4 kb
4 kb
8 Kb
3 kb
3 kb
9 kb
5
12
6 kb
11 kb
6 kb
5 kb
1 kb
3 kb
3kb
9 kb
4 kb
4 kb
8Kb
6
8
1 kb
6 kb
1 kb
5 kb
2 kb
2 kb
5 kb
2 kb
3 Kb
3 Kb
3 kb
4 kb
3 kb
1 kb
4 kb
Gel electrophoresis:
Agarose gel shows us 5 wells (1st well with A restriction enzyme. 2 well with N restriction enzyme, 3rd well with A+N restriction enzyme, 4rth well with E restriction enzyme and 5th well with restriction enzyme).
And on the Y axis we can see the Fragment size in Kb. This shows the size of the fragments formed after restriction.
We will study all the wells:
Well
Restriction enzyme
A
N
A+N
E
E+N
cleaves the DNA at
Fragments formed
cleaves the DNA at
Fragments formed
cleaves the DNA at
Fragments formed
cleaves the DNA at
Fragments formed
cleaves the DNA at
Fragments formed
1
2 sites
3 kb
9 kb
2
2 sites
4 kb
8 kb
3
3 sites
1 Kb
3 kb
8 kb
4
3 sites
1 Kb
5 kb
6 kb
5
4 sites
1 kb
2 kb
4 kb
5 kb
5.
a. Which of the six restriction maps shown below is consistent with the observed pattern of restriction fragments?
Ans: Thus clearly shows it restriction map 5 is consistent with the observed pattern of restriction fragments. We can come to conclusion after checking all the fragments formed which are clearly shown in both the tables as bolded.
B. Where is the pep gene located in the correct restriction map?
Ans:, Examining all those locations of pep gene in the restriction map 5, Pep must be in between 3 -4 kb fragment size positions
Fragment 5: ........................A3 kb.......N 4kb..................E.............................E
Take well 1 which gives us 9 Kb fragment with A, which shows (12kb= 3+9kb) the gene should be at 2nd fragment side.
2 Well we can see 4 kb length fragment with N, which shows (12kb= 4kb+8 kb) the gene should be at 1st fragment side.
From this we can say our required pep gene in the restriction map 5 is in between 3 to 4 kb length
Further 3rd wll, A+N digestion gives us 1 kb (3to 4 kb region). so this confirms our PEP is a 1kb length fragment between 3-4 kb length
Well
Restriction enzyme
A
N
A+N
E
E+N
Fragments formed
Fragments formed
Fragments formed
Fragments formed
Fragments formed
1
9 kb
2
4 kb
3
1 kb
4
6 kb
5
4 kb
Restriction map in order from top to bottom
Length Kb
E restriction enzyme
A restriction enzyme
N restriction enzyme
Cleaves at
Fragments size
Cleaves at
Fragments size
Cleaves at
Fragments size
1
12
6kb
11 kb
6 kb
5kb
1 kb
8kb
8 kb
4 kb
3 kb
3 kb
9 kb
2
12
6 kb
11 kb
6 kb
5 kb
1 kb
3 kb
3 kb
9 kb
8 kb
8 kb
4 kb
3
12
6 kb
11 kb
6 kb
5 kb
1 kb
8kb
8 kb
4 kb
9 kb
9 kb
3 Kb
4
12
6 kb
11 kb
6 kb
5 kb
1 kb
4 kb
4 kb
8 Kb
3 kb
3 kb
9 kb
5
12
6 kb
11 kb
6 kb
5 kb
1 kb
3 kb
3kb
9 kb
4 kb
4 kb
8Kb
6
8
1 kb
6 kb
1 kb
5 kb
2 kb
2 kb
5 kb
2 kb
3 Kb
3 Kb
3 kb
4 kb
3 kb
1 kb
4 kb