question about sanger sequencing (1 Viewer)

[drm]

if sanger sequencing involves the random addition of ddNTPs, then how can it be guaranteed that every single position of a particular base (A, C, T, G) is marked by the ddNTP?

 

[drm]

if sanger sequencing involves the random addition of ddNTPs, then how can it be guaranteed that every single position of a particular base (A, C, T, G) is marked by the ddNTP?

bump

 

[Eagle Mum]

if sanger sequencing involves the random addition of ddNTPs, then how can it be guaranteed that every single position of a particular base (A, C, T, G) is marked by the ddNTP?

DNA is amplified before the sequencing step. Thirty cycles produce about a billion DNA templates, so for example, if the ddNTP:dNTP ratio is 1:1000, then a million fragments would be the ballpark number for each terminating ddNTP representing any particular position, so the probability that a base is missed is extremely low (though not impossible) and vanishingly small that the same base is missed in both the forward and reverse sequencing reactions.

The ratio of ddNTP to dNTP in the reaction mixture also needs to be optimised since it determines the distribution of DNA fragment lengths produced. Sanger sequencing runs do sometimes fail if the ratio isn’t optimised. True DNA variants (ie. mutations & polymorphisms) should be present in both the forward and reverse sequence, otherwise it’s likely to be a sequencing error of the type you are suggesting could/would happen.