Sequencing by the Sanger Dideoxynucleotide Chain Termination Method 1. Prepare replication template...
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Sequencing by the Sanger Dideoxynucleotide Chain Termination Method 1. Prepare replication template denature, add synthetic primer, promote annealing TAGGCGA GATCTG 5’ 3’ CTAGAC 5’ 3’ TAGGCGA GATCTG 5’ 3’ unknown sequence DNA template known sequence 5’ 3’ Add components for in vitro replication to each tube, add: - reaction buffer (salts, pH, etc.) - 4 dNTP precursors (1 radioactively labeled) - DNA polymerase ddCTP ddTTP ddGTP ddATP tube with large number of annealed DNA and primer complexes from step 1 distribute into 4 separate tubes 1 dideoxynucleotid e ddATP H
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Transcript of Sequencing by the Sanger Dideoxynucleotide Chain Termination Method 1. Prepare replication template...
Sequencing by the Sanger Dideoxynucleotide Chain Termination Method
1. Prepare replication template
denature, add synthetic primer,promote annealing
TAGGCGA GATCTG5’ 3’CTAGAC
5’3’
TAGGCGA GATCTG5’ 3’
unknown sequence
DNA template
known sequence
5’3’
2. Add components for in vitro replication
to each tube, add:- reaction buffer (salts, pH, etc.) - 4 dNTP precursors (1 radioactively labeled)- DNA polymerase
ddCTP ddTTP
ddGTP
ddATP
tube with large number of annealed DNA and primer complexes from step 1
distribute into 4 separate tubes
1 dideoxynucleotide
ddATP
H
Sequencing by the Sanger Dideoxynucleotide Chain Termination Method, continued
4. Electrophoresis and visualization of replication products
TAGGCGA GATCTG5’ 3’CTAGAC
5’3’
3. Replication reactions
CTAGAC
CTAGACCTAGAC
5’5’5’
CTAGACCTAGAC5’
5’
CTAGAC5’
CTAGAC5’
{{{
{
ddCTP
ddTTP
ddGTP
ddATP
ddC rxn
ddT rxn
ddG rxn
ddA rxn
+
-
T
G
CC
TA
C
5’
3’
A
C
GG
AT
G
5’
3’
Automated DNA Sequencing
Use 4 ddNTPs, each labeled with different fluorochrome, in single reaction.
Individual products are differentiable by size and attached fluorochrome.
As bands migrate during electrophoresis, a detector senses the different fluorochromes and feeds the information to a computer for data analysis.
