2007-2008

A Lot More Advanced Biotechnology Tools(Part 1)

Sequencing

• Sanger method– determine the base sequence of DNA– based on replication– dideoxynucleotides• ddATP, ddGTP, ddTTP, ddCTP• missing O for bonding of next

nucleotide• terminates the growing chain

DNA Sequencing

DNA Sequencing• Sanger method– synthesize

complementary DNA strand in vitro

– in each tube:• “normal” N-bases• dideoxy N-bases

– ddA, ddC, ddG, ddT

• DNA polymerase• primer• buffers & salt

2

1

3

42

Reading the sequence• Load gel with sequences from ddA, ddT,

ddC, ddG in separate lanes– read lanes manually & carefully– polyacrylamide gel

Fred Sanger 1978 | 1980

This was his 2nd Nobel Prize!!– 1st was in 1958 for the structure of

insulin

Advancements to sequencing• Fluorescent tagging– no more radioactivity– all 4 bases in 1 lane• each base a different color

• Automated reading

Advancements to sequencing• Fluorescent tagging sequence data• Computer read & analyzed

Applied Biosystems, Inc (ABI) built an industry on these machines

Advancements to sequencing Capillary tube electrophoresis

no more pouring gels higher capacity & faster

384 lanes

PUBLIC Joint Genome Institute

(DOE) MIT Washington University of St.

Louis Baylor College of Medicine Sanger Center (UK)PRIVATE Celera Genomics

Big labs! economy of scale

Automated Sequencing machines Really BIG labs!

Human Genome Project• U.S government project– begun in 1990

• estimated to be a 15 year project– DOE & NIH

• initiated by Jim Watson• led by Francis Collins

– goal was to sequence entire human genome• 3 billion base pairs

• Celera Genomics– Craig Venter challenged gov’t – would do it faster, cheaper– private company

Different approaches

3. Assemble DNA sequence using overlapping sequences.

“map-based method”gov’t method

“shotgun method”Craig Venter’s method

1. Cut DNA entire chromosome into small fragments and clone.

2. Sequence each segment & arrange based on overlapping nucleotide sequences.

1. Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments.

2. Cut and clone into smaller fragments.