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Biochem Brandl 11

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Western University
Biochemistry 2280A
Eric Ball

Biochem - Lecture 11 - Dec. 1st Sequencing Genomes: • Craig Ventor vs. Francis Collins (racing to sequence the genome, ended up work- ing together to get it done faster) • The Language of Life: DNA and the Revolution In Personalized Medicine - Fran- cis Collins Sequencing a Genome Involves: • creating a Genomic DNA Library • many independent sequencing reactions • aligning the independent sequences into a continuous sequence Genomic Library: • definition: a collection of cloned DNA fragments that represent all of the DNA in an organism’s genome • each cloned DNA is like a different book Constructing a Genomic Library: • Figure 10.11 • human DNA is cleaved with restriction nuclease to create millions of genomic DNA fragments • fragments are inserted into plasmids using ligase • recombinants DNA molecules are introduced (as plasmids) into bacteria Sequencing the H. Influenza Genome: • gram negative bacteria • genome is approximately 2 million bp • Question: If the genome is 2 million bp, how many independent clones must be in the genomic library to cover the genome at least once, assuming an average in- sert size of 2000 bp? • Answer: 1000 - would be the absolute minimum, to be sure you have ful coverage, you would want an excess of several fold • extracted DNA from the bacteria, then sonicated it (more random than restriction enzymes) to create fragments • used agarose gell electrophoresis to seperate fragments and removed those that were around 2000 bp • purified DNA from the gel (DNA fragments 1.6-2.0 kb) and prepared a genomic li- brary • 20,000 clones - each represents an independent fragments of the genome, 20- fold coverage Sequencing the Ends of All 20,000 Genomic Clones: • isolate plasmid DNA • anneal primer • dideoxy sequence • 25,000 sequ
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