3.16 - DNA Sequencing with the Sanger Dideoxy Method

Question 1

What is the “scientific name” for free nucleotides?
What are they used by?

Answer 1

deoxynucleotide triphosphate dNTP
They are used by TAQ polyermase

Question 2

What are ddNTPs?
What is its nickname?

Answer 2

Dideoxynucleotide triphosphate
Called a “broken [base] triphosphate”

Question 3

What’s the difference between dNTP and ddNTP?

Answer 3

ddNTPs are missing the OH group on the 2nd and 3rd carbons on the sugar molecue, while dNTP only miss an OH on the 2nd carbon.

Question 4

What happens when ddNTP is added to a sequence?

Answer 4

They sequence stops.

Question 5

What happens as many rounds of PCR testing are performed?

Answer 5

-As rounds of PCR are done, with the addition of ddNTPs, eventually all possible strands will be created
Ex) when ddATP is added, it’ll stop at all the adenines in the sequence.This occurs for each DNA base

Question 6

Is the Sanger sequencing method automated?

Answer 6

Yes.
It is computer run

Question 7

Compare the old way of using the Sanger Dideoxy method with the new way.

Answer 7

Old: - required a 4 line gel (one for each base) & no colours were assoiated with each base.
New: - ddNTPs are mixed with different colour dyes to tell them apart, meaning they can now be done on single lines gels. it’s also computer automated as the can scan the colours

Question 8

How do you read an old style Sanger Dideoxy method gel?
What end do you start reading the sequence at?

Answer 8

• Start from the bottom of the gel (+), and work your way up
• whichever base has the least number of base pairs to the second least, and so on.
• • Ex) if cytosine has 5 fragments, then guanine has 6, and adenine has 7, the begining of the sequence would look like CGA…
• you read/identify the sequence starting at the 5’ end to the 3’ end