04 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 wit

h 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