Transcript DNA Sequencing

DNA Sequencing
DNA sequencing
…ACGTGACTGAGGACCGTG
CGACTGAGACTGACTGGGT
CTAGCTAGACTACGTTTTA
TATATATATACGTCGTCGT
ACTGATGACTAGATTACAG
ACTGATTTAGATACCTGAC
TGATTTTAAAAAAATATT…
DNA sequencing
 Determination of nucleotide sequence
 Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
 They depend on the production of a mixture of
oligonucleotides labeled either radioactively or
fluorescein, with one common end and differing in
length by a single nucleotide at the other end
 This mixture of oligonucleotides is separated by high
resolution electrophoresis on polyacrilamide gels
and the position of the bands determined
Maxam and Gilbert Method
The single stranded DNA fragment to be sequenced is endlabeled by treatment with alkaline phosphatase to remove the
5’phosphate
It is then followed by reaction with P-labeled ATP in the
presence of polynucleotide kinase, which attaches P labeled to
the 5’terminal
The labeled DNA fragment is then divided into four aliquots,
each of which is treated with a reagent which modifies a
specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue
2. Aliquot B + formic acid, which modifies adenine and guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for
cytosine
The four are incubated with piperidine which cleaves the sugar
phosphate backbone of DNA next to the residue that has been
modified
Frederick Sanger
• Discovered DNA sequencing by chain
termination method
• Nobel Prize 1 (1958)
– Complete amino acid
sequence of insulin
• Nobel Prize 2 (1980)
– For DNA sequencing
Sanger Method
 DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
 Requires a primer, DNA polymerase, a template, a mixture
of nucleotides, and detection system
 Incorporation of dideoxynucleotides into growing strand
terminates synthesis
 Synthesized strand sizes are determined for each
dideoxynucleotide rxn by using gel or capillary
electrophoresis
Dideoxynucleotide
PPP
O
5’
CH2
O
BASE
3’
no hydroxyl group at 3’ end
prevents strand extension
Dideoxy nucleotides
• Incorporation of a dideoxynucleotide to
growing DNA strand terminates its further
extension
• Are added in small proportion
–
–
–
–
dATP
dGTP
dCTP
dTTP
ddATP
ddGTP
ddCTP
ddTTP
Chain Termination
Chain Terminator Basics
Target
Template-Primer
TGCA
ddA
Extend
ddA
AddC
AC ddG
ACG ddT
ddT
ddC
ddG
Labeled Terminators
dN : ddN
100 : 1
3’
primer 5’
CCGTAC 5’
3’
dNTP
ddATP ddTTP
GGCA
ddCTP ddGTP
GGCAT
A
T C
GGC
G
G
GG
GGCATG
All Possible Terminations
Polyacrylamide Gel Electrophoresis
Separates
fragments
based on size
Electrophoresis
DNA Sequencing – vectors
DNA
Shake
DNA fragments
Vector
Circular genome
(bacterium, plasmid)
+
=
Known
location
(restriction
site)
Different types of vectors
VECTOR
Size of insert
Plasmid
2,000-10,000
Can control the
size
Cosmid
40,000
BAC (Bacterial Artificial
Chromosome)
70,000-300,000
YAC (Yeast Artificial
Chromosome)
> 300,000
Not used much
recently
DNA Sequencing – gel
electrophoresis
1.
2.
3.
4.
5.
Start at primer
(restriction site)
Grow DNA chain
Include
dideoxynucleoside
(modified a, c, g, t)
Stops reaction at all
possible points
Separate products with
length, using gel
electrophoresis
Template
• ssDNA vectors
– M13
– pUC
• PCR
• dsDNA (+/- PCR)
Primers
• Universal primers
– cheap, reliable, easy, fast, parallel
– BULK sequencing
• Custom primers
– expensive, slow, one-at-a-time
– ADAPTABLE
Extension Chemistry
• Polymerase
– Sequenase
– Thermostable (Cycle Sequencing)
• Terminators
– Dye labels (“Big Dye”)
• spectrally different, high fluorescence
– ddA,C,G,T with primer labels
Separation
• Gel Electrophoresis
• Capillary Electrophoresis
– suited to automation
• rapid (2 hrs vs 12 hrs)
• re-usable
• simple temperature control
• 96 well format
Sequencing Strategies
• Ordered
– Divide and Conquer
• Random Sequence
– Brute Force
DNA Sequencing 5.17)
Sequencing of DNA by
the Sanger method