Molecular Biology: DNA sequencing
Molecular Biology:
DNA sequencing
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.
SEQUENCING BY DIDEOXY CHAIN TERMINATION
The sequencing method most commonly used today is the dideoxy chain-termination method (Sanger
et al., 1977). Dideoxy sequencing allows the determination of 200 to 1000 nucleotides from a singlestranded DNA template, using as starting material either cloned or amplified DNA.
The dideoxy sequencing reaction mix includes the template DNA, deoxynucleoside
triphosphates (dNTPs), dideoxynucleoside triphosphates (ddNTPs), a DNA
polymerase enzyme and a primer.
Like the Polymerase Chain Reaction (PCR), dideoxy sequencing starts with the denaturation of the
DNA template and the binding of an oligonucleotide primer (annealing), but unlike PCR a single
primer is used. (For a detailed description of PCR, see the PCR techniques course notes.) DNA
polymerases require the presence of a small piece of DNA, the primer, in order to initiate the growth
of a DNA chain. The primer is complementary to the DNA template and chain elongation starts at the
3’ end of the primer. The primer therefore determines the starting point of the sequence being read,
and the direction of the sequencing reaction. For sequencing of the insert of a plasmid template, a
sequencing primer based on the vector sequence adjacent to the insert can be used. For PCR
products, either the forward or reverse amplification primer can be used as the sequencing primer, or
an internal sequencing primer can be designed.
One of the single most important factors in successful DNA sequencing is proper PRIMER DESIGN.
A sequencing primer should have the following characteristics:

A melting temperature (Tm) in the range of 52°C to 65°C

No internal secondary structure

Balanced distribution of G/C and A/T rich domains

No complementarity between 3’ ends (so primer-dimers will not form)

No significant hairpin formation (>3 bp) (if there are regions of self-complementarity in the primer
sequence, the primer can anneal to itself and fold up to form a hairpin loop)


Lack of secondary priming sites in the target sequence
Low specific binding at the 3' end (i.e. lower GC content to avoid mispriming)
Starting from the 3’ end of the annealed primer, the DNA polymerase synthesizes a new DNA strand,
adding dNTPs or ddNTPs that are complementary to those on the template. If allowed to go to
completion, a new strand of DNA would be the result. However, ddNTPs have no 3' hydroxyl (3'-OH)
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Molecular Biology: DNA sequencing
group (see Figure 2), so once a ddNTP is incorporated at the end of a growing DNA strand, there is
no way for the polymerase to continue elongating it. The dNTP:ddNTP ratio is adjusted so that most
of the time the polymerase enzyme will use a dNTP, but every so often a ddNTP will be chosen,
which will terminate the elongation reaction.
At the end of the sequencing reaction, there are multiple copies of every possible fragment, each
terminated by a ddNTP. Separation of the reaction products on long, thin, high-resolution
polyacrylamide gels (sequencing gels) or in thin capillaries containing liquid polymer allows the
determination of the nucleotide sequence.
Figure 1: Dideoxynucleoside triphosphates (ddNTPs) lack a 3' hydroxyl group, and if incorporated into a
growing strand of DNA, they will terminate the elongation reaction.
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