DNA sequencing
The first generationThe Sanger dideoxy method of DNA sequencing
The chain termination method
The Sanger dideoxy method of DNA sequencing
This method was costly, at around $10 per base pair in 1985, but the development of automated sequencing
systems and advancements in technology reduced the price to $1 per base by 1995, and allowed sequencing of up to 100,000 bases per day. The cost dropped further to $0.10 per base in 1998
with the development of the ABI Prism sequencer, which made it possible toundertake larger-scale sequencing projects.
• In the context of highthroughput shotgun genomic sequencing, Sanger sequencing costs on the order of $0.50 per kilobase.
• National Institutes of Health (NIH), specifically the NIH’s National Human Genome Research Institute (NHGRI), has actively sponsored researchers with $99.5 million of external funding to develop sequencing technologies since the completion of the Human Genome Project.
• $100,000 initially and then as little as $1000.
In search of the $1000 genome
The second generation: sequencing-by-synthesis
• Pyrosequencing ($1.5million, 2008) • Illumina ($48,000 ?, 2010)
• SOLiD
pyrosequencing
Enzyme mix 5ulSubstrare mixture 5ul100pmol primer 1ul500fmol template 5ul10xBuffer 10ul水 74ul
control
0
200000
400000
600000
800000
1000000
1200000
ES A G T C(108)
(58,632)
(47,684)
(244,154)
196,470
(163,395)
969,984
(1,133,379)
(199,549)
54,249 (253,79
8)
(904,103)
667,372
(236,731)
(253,619)
GCAGGATG
間隔 2min dNTP 0.2x
Roche/454 FLX Pyrosequencer
The third generation:direct measuremeant or synthesis-free
• Nanopore
Nanopore detector