DNA sequencing. How DNA synthesis takes place inside the living cell?
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Transcript of DNA sequencing. How DNA synthesis takes place inside the living cell?
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DNA sequencing
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How DNA synthesis takes place inside the living cell?
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Can you think of a way to determine the DNA seq. in vitro ?
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The idea of DNA sequencing
The use of ddNTPs + the normal dNTPs
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There are 2 methods for DNA sequencing
1 -Classic sequencingUsing radioactivity
2 -Modern sequencingUsing fluorescence
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The classic sequencing
Sanger Chain termination method
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Modern sequencing
Chain termination method
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Steps for DNA sequencing
1 -DNA isolation and purification2 -PCR
3 -Amplified DNA purification 4 -Termination 5 -Sequencing
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1 -DNA isolation and purification
Obtain high quality DNA to be sequenced1 -Conc. by spec *Calculation.……
2 -Integration
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2 -Is PCR for sequencing different from the general PCR
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3 -Amplified DNA purification
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General PCR components
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General PCR strategy
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PCR Program
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PCR objective
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PCR for sequencing
Components of PCRTemplate + terminator ready reaction mix .
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The PCR program
Hot start 2 minDen. 10 sec @ 94oCAnnealing 5 sec @ 50 oCExtension 4 min. @ 60 oC
25 cycles
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Seq. PCR end product
•What are the characteristics of the resulted DNA?
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How to handle the DNA after this point
Purification Termination
Sequencing
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What happens inside the sequencer
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Review over the sequencing process
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