Comparison between old generation and new generation of

sequencing machines

Review over the sequencing process

Genome sequencing projectHuman genome

Genome sequencing project

•Shot gun method

•DNA library

•Sequencing

•Assembly

Efficiency of old generation of DNA sequencer

•How many bases it can sequence and how long it takes?

Now with new generation

•You can sequence bacterial genome in less than a day

•Arabidopsis genome in days

•Human genome in less than 3 weeks

Prepare the library

Nebulizing the DNA

Nebulizing the DNA

•DNA is fragmented into small sizes

•50 bp-1Kbp

•Is it a wide range??????

How to select a narrower DNA size?

DNA size selection

Select from 500-800 bp

Repair purified nebulized DNAby end polishing

Three enzymes are used :

Extend by..………………

Remove by………………

Capping by.………………

Adaptor ligation

A : Green

B: Red and biotinylated

After Adaptor ligation

•4 products are produced

DNA immobilization

•Biotin::striptavidin interaction•Which product will be washed away ?•Which product

Will be sequenced

or non sequenced

ss DNA library formation

Predict what will happen if we add NaOH soln ? .

What you will have in the supernatant?

The final ssDNA library

PCR of DNA

Mixing the beads and DNA

•Beads contains on their surface…………

•The beads and ssDNA are mixed in

…………………PCR reaction results in

..……………Microreactors contain

………………………. Mix .

PCR preparation

4 Of 96 wells micro titter plate

DNA amplification

DNA orientation on the beads

•The beads contain DNA coplementary to …………………. Side .

•The B adaptor will be ……….., the A adaptor will be……………

Isolation of the positive amplified DNA

Separation of amplified DNA

•Magnetic particle concentrator

Sequencing reaction Another PCR

After this sequencing the beads will be added onto the slide

Titanium slide

One bead goes in one well on the titanium slide and then this slide will

be placed for sequencing