LT7 DNA Sequencing

DNA Sequencing

 Sanger method most widely used – dideoxy DNA sequencing, method based on
DNA synthesis carried out in vitro in the presence of special, chain-terminating
dideoxyribonucleoside triphosphates
 DNA polymerase is used to make partial copies
of the DNA fragment to be sequenced
 DNA replication reactions performed under
conditions that ensure the new DNA strands
terminate when a give nucleotide (A,G,C,T) is
reached – reactions ultimately produce a
collection of different DNA copies that
terminate at every position in the original
DNA, and thus differ in length by a single
nucleotide
 DNA copies can be separated on the basis of
their length by gel electrophoresis, and the
nucleotide sequence of the original DNA can be determined from the order of
the DNA fragments in the gel

The Process

- The reaction is carried out in
parallel with each of the
dideoxynucleotides (ie. 4 reactions
– ddAGP, ddGTP, ddCTP and ddTTP)
- The reaction products are run in
parallel on a sequencing gel and
visualised by autoradiography –
insepection of the pattern of bands allows the complete sequence to be
reconstructed
1. To determine the complete sequence of a DNA fragment, the ds DNA
denatured, one of the ssDNA strands becomes template for sequencing
2. Four different dideoxynucleoside triphosphates used in 4 separate DNA
synthesis reactions on copies of the same ssDNA template
3. Each reaction produces a set of DNA copies that terminate at different
points in the sequence
4. Products of 4 reactions separated by electrophoresis in 4 parallel lanes of a
polyacrylamide gel