DNA Sequencing

  • #1
For sequencing using the Sanger model, what types of bands on an electrophoresis gel would be produced if two different ddNTPs instead of just one ddNTP were added to a tube of DNA polymerase, template, and dNTPs? Would it be correct to think of which bands would be produced by each ddNTP independently of the other, and, then, these bands from each different ddNTP can be collectively represented on the gel?

Thank you.
 
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Answers and Replies

  • #2
Ygggdrasil
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Yes. The bands in the lane with the two ddNTPs would basically just be the combination of band from ddNTP 1 and bands from ddNTP 2.
 
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  • #3
Do you mean "ddNTPS" (in place of where you typed "dNTPS")?

Thank you again.
 
  • #4
Ygggdrasil
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Yes, I meant ddNTPs. I've fixed the post. Thanks.
 
  • #5
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Yes, but keep in mind that this method is rather antiquated and really no longer used. These days, the reactions are all done in a single tube with dye-labeled nucleotides.
 
  • #6
bobze
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Yes, but keep in mind that this method is rather antiquated and really no longer used. These days, the reactions are all done in a single tube with dye-labeled nucleotides.
This is true, the way is paved by high-throughput sequencers :smile: That doesn't mean that students still don't have to learn more "traditional" techniques!
 
  • #7
I have a question regarding direction of a DNA strand and sequencing. Suppose I have the following imaginary DNA fragment (which could actually exist in a species but I do not really know). This fragment was sequenced by the Sanger method.

GATTACCCAGCCTAATTC

A primer with a labelled 5' end is introduced, and it binds to the first eight bases. (I do not know if the primer length is feasible or if it is too short. I am just attempting to set up a hypothetical example.)

What exactly is the direction of the above DNA fragment? Normally, when I see a DNA sequence, the left end is the 5' side and the right is the 3' side. However, since the above fragment was sequenced by the Sanger method, is the direction really reflective of the complementary strand's direction, 3' to 5'? in other words, the above fragment is not the template, and, therefore, it is not in the 5' to 3' direction?

Thank you.
 
  • #8
bobze
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If you were looking at it on a gel, the lowest bands would correspond to the 5' end. Which should make sense if you think about it as they would be the shortest and migrate the farthest on the gel (Remember DNA poly can only add to the free 3'-OH, so synthesis must go 5'→3', so the first nucleotide added to the primer will be the shortest band). So if your hypothetical is written in the 5'-3' direct, on a gel it would actually look like;

3'
C
T
T
A
A
T
C
C
G
A
C
C
C
A
T
T
A
G
5'


Also make sure you are careful to know if you've sequenced the template or coding strand. As we typically want to write the coding strand, left to right (5'-3').

(Edit: although remember they would be spread out in 4 lanes, so it wouldn't be a straight line like that)
 

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