DNA sequenced. Erased labels. Now re-identify.

[doctobe]

Hello. So I have a homework question that essentially goes like this.

I extracted DNA containing the same gene from two individiuals then sequenced it using the dideoxy method.
I was able to get there sequenced based on the autoradiodiagram.

However, I erased the identifying lables on the tubes from which I extracted the DNA. Sequence A and B have the same sequences except for the four base pairs.
(5"- 3′)
A ACTGAATTCCGATG
B ACTTAATTCCGATG

I was given three sets of restriction enzymes.(Ecor1 "5-GAATTC-3′), (PvuI "5-CGATCG’3) and (Tsp5091 5′-AATT-3′)

The question asks to "Explain how you would re–identify the tubes (A and B) without performing another sequencing reaction.

My hypothesis. Is I cut with the Tsp5091 restriction enzyme. . THis would leave me with two DNA strands. one with (5′- ACTG-3′) and another with (5’=ACTT-3′) Right? Then what? I do re-identify the tubes?

[JackBean]

it would be easier, if you used the letters 🙄 And I see only one difference

Also, you should mention, what for DNA is that. Is it gDNA, vector or some amplicon?

[doctobe]

I edited it. Easier?

[JackBean]

better, but wrong.
Notice, that in A you have 5-GAATTC-3, whereas in B you have 5-TAATTC-3, so the A-DNA will be cut one more time with EcoRI and thus the pattern on gel will be different.
But the question is still, what kind of DNA you have. If amplicon or short vector, than it’s OK, if gDNA, than you had to perform blotting…

[doctobe]

Okay I think I understand it. Tsp for the B strand and Eco R1 for the A strand. Then do a southern blot hybridization since it I believe it is a gDNA I believe. I pmed you for more detail and clarification.

[JackBean]

No, no, use the same RE (EcoRI) for both (you don’t know, which one is which one;).
Than you should do probe for your part of DNA and perform hybridization.

[doctobe]

So do you you always use one? I don’t know which one is which but I know the sequence right?

[JackBean]

You can use more, of course (although more than two are not used much probably), but you have to check where will it cut around.
But you should not use EcoRI and Tsp at once! Because than you would got two bands in both cases.

[doctobe]

OH. I thought we wanted two bands. But usually want one? that way when we identify one we automatically know the other one?

[JackBean]

No, you NEED to see some difference. In this case, in tube with ACTGAATTCCGATG sequence, you will get two smaller bands on blot after digestion with EcoRI, whereas with the sequence ACTTAATTCCGATG you will get only one larger.

You have some chromosome and on it you have this sequence, let´s say, you will prepare probe about 100 bp upstream and 100 bp downstream.

Each restriction enzyme cut with some probability after several (hundreds/thousands) of bp, you can calculate that easilly or find somewhere. So, you can either find, where exactly EcoRI cuts, if you know the sequence or just expect the result.
Anyway. In the case of B, EcoRI will cut somewhere outside on both sides and thus your probe will bind only to one strand (meaning several strands of one size;), whereas in the case of A, this one strand will be cut "in half" and thus producing two strands.

Of course, EcoRI can cut closer, than your probe boundaries will be, but you will always get n and n-1 bands 😉

[doctobe]

I need to do some more reading on restriction. I know it is simple but I need to be confident.

[doctobe]

quote JackBean:

You can use more, of course (although more than two are not used much probably), but you have to check where will it cut around.
But you should not use EcoRI and Tsp at once! Because than you would got two bands in both cases.

Okay So I could just cut one sequence with ECO R1 and then do a southern blot hybridization.

I understand the concept of cutting. But I don’t understand the concept of cutting multiple times. I know that there the strand is longer than the sequence given. Since I only know that sequence though isn’t that enough. If I find one that I automatically know the other strand label?

how can I use Eco R1 for both strands.?

Strand B doesn’t have a sequence for EcoR1. It has a sequence for (Tsp5091 5′-AATT-3′)

[JackBean]

The DNA is not long only ~10 nt, it’s long at least several thousands or millions of bp, so the EcoRI cut place is there somewhere, as I told you. Also, as I told you, you need to distinguish between both samples and THUS you need to once cut and once not to cut!

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