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TECHNOLOGY
Dr.M.Vinoth
Animal biotechnology
Adapted from Eric Green, NIH; Adapted from Messing & Llaca, PNAS (1998)
History of DNA Sequencing
50,0 1986
00
• Cycle Sequencing
200,0 1990 • Improved Sequencing Enzymes
00 • Improved Fluorescent Detection Schemes
50,000,00
0 2002
• Next Generation Sequencing
•Improved enzymes and chemistry
100,000,000,000 2009
•New image processing
What is need of
sequencing ?
• Fundamental requirement in biotech
• Disease genotyping-identify specific genetic
diseae(birth itself)-can give gene therapy
• Organism genome sequencing-screening and
further study about organism
• Research-primer designing, in genomics,
transcriptomics, and epigenomics etc
• Diagnostic tests,rDNA tech
Basics of the “old” technology
sequencers
100 Gb
AB/SOLiDv3, Illumina/GAII
short-read sequencers
(10+Gb in 50-100 bp reads,
10 Gb
$1000 ,
bases per machine run
10 bp 100 bp 1,000 bp
read length
Helicosbio
• Helicos True Single Molecule Sequencing
(tSMS)™
• Eliminating the need for amplification and
its associated biases and error
• billions of single molecules of sample DNA
are captured on an application-specific
proprietary surface.
• Polymerase and one fluorescently labeled
nucleotide (C, G, A or T) are added.
• The polymerase catalyzes the
sequence-specific incorporation of
fluorescent nucleotides into templates.
• After a wash step,the incorporated
nucleotides are imaged and their
positions recorded.
• Then fluorescent group is removed.
Then next nucleotide incorporated .
• images billions of single molecules
per run and produces over one
gigabase of usable sequence data
per day
• throughput performance- 100X
greater than Sanger methods
• Whole genome-around $1000
pacificbiosciences
• Instead of inspecting DNA copies after
polymerase
• Single Molecule Real-Time (SMRT)
sequencing watches the enzyme in real
time
• DNA Polymerase attach on the surfase of
tiny well
• Every nucleotide fluorescently label with
different colour when it incorporate emit
different wave length light
pacificbiosciences
pacificbiosciences
• Long read,fast results low overall
cost
• now they sequencing genome for
$1000
$100 genome
• could enable
sequencing in 15 minutes in
2013.
REFERENCE
• http://454.com/
• http://illumina.com/
• http://appliedbiosystems.com/
• http://pacificbiosciences.com/
• http://helicosbio.com