DNA sequencing

How DNA synthesis takes place inside the living cell?

Can you think of a way to determine the DNA seq. in vitro ?

The idea of DNA sequencing

The use of ddNTPs + the normal dNTPs

There are 2 methods for DNA sequencing

1 -Classic sequencingUsing radioactivity

2 -Modern sequencingUsing fluorescence

The classic sequencing

Sanger Chain termination method

Modern sequencing

Chain termination method

Steps for DNA sequencing

1 -DNA isolation and purification2 -PCR

3 -Amplified DNA purification 4 -Termination 5 -Sequencing

1 -DNA isolation and purification

Obtain high quality DNA to be sequenced1 -Conc. by spec *Calculation.……

2 -Integration

2 -Is PCR for sequencing different from the general PCR

3 -Amplified DNA purification

General PCR components

General PCR strategy

PCR Program

PCR objective

PCR for sequencing

Components of PCRTemplate + terminator ready reaction mix .

The PCR program

Hot start 2 minDen. 10 sec @ 94oCAnnealing 5 sec @ 50 oCExtension 4 min. @ 60 oC

25 cycles

Seq. PCR end product

•What are the characteristics of the resulted DNA?

How to handle the DNA after this point

Purification Termination

Sequencing

What happens inside the sequencer

Review over the sequencing process