Download DNA Sequencing

DNA Sequencing
Biotechnology II
Essential Question
What technology is used to determine the
sequence of DNA?
DNA Sequencing
 Once DNA amplified, its
nucleotide sequence
determined
 Today, DNA sequencing is
automated
 Automated system
originally based on
Dideoxy Chain
Termination Method
 Invented by Frederick
Sanger - received Nobel
Prize in 1980
Deoxy Chain Termination Method
 Based on DNA replication
 Makes use of
dideoxyribonucleotides
 Remember deoxyribose found
in DNA– missing an oxygen at 2
carbon
 Dideoxyribose – missing two
oxygens – at 2 and 3 carbon
 Significance:
 Oxygen at 3rd Carbon required
for DNA strand extension
 Where free nucleotide attached
 When dideoxyribose
incorporated into DNA
molecule, DNA extension stops
DNA Sequencing
Reaction Mixture
 Gene to be sequenced
denatured and incubated in
test tube with
• primer complementary to 3’
end of gene,
• DNA polymerase,
• four deoxyribonucleotides
• four dideoxyribonucleotides
tagged with radiolabel 32P
DNA Sequencing
Steps
 Synthesis of new strand
starts at 3’ end of primer
and continues until
dideoxyribonucleotide is
inserted- stops synthesis
 Separate labeled strands
through a polyacrylamide
gel
 Placed on x-ray film
 Because of florescent tag,
different length fragments
are revealed on film and
read manually
Example of DNA sequence
Problem with Sanger’s Method
Labor intensive
Took a long time to
sequence a gene
that was fairly large
in size (1000-2000
bp)
Often times
mistakes made in
interpreting gels
Advancement in Technology
 First major advancement
came with the creation of
florescent tags for
dideoxyribonucleotides
rather than radiolabel
 Each dideoxy base given a
different color tag
 Example




Adenine – green
Thymine –red
Guanine – yellow
Cytosine -Blue
 Florescent label can now be
read with laser detector
eliminating radiolabel and xray film
Advancement in Technology
 Eventually, the whole
process became automated
 Contain detector systems
that detect the florescent
dye
Class Activity
Get into groups and complete the
simulation on the dideoxy method initially
developed by Sanger