Working with Genomes – Chapters 16 & 17
Restriction Endonucleases
– Type I - simple cuts
– Type II - dyad symmetry
 allows physical mapping
 allows recombinant molecules

Because of complementarity, single-stranded ends can pair with each other.
 sticky ends
 fragments joined together with DNA ligase
Restriction Endonucleases Video
Notes:
Host / Vector Systems
Using Vectors to Transfer Genes
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Chimeras
– One of first recombinant genomes was a bacterial plasmid into _______________
_____________________________________.
 Viruses can also be used as ___________________________ into host cells.
Early Genetic Engineering
Genetic Engineering Video Notes
DNA Libraries
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A collection of DNA from a ___________________________________________________
– genomic library - ______________________________________________________
– cDNA library is limited to expressed genes __________________________________
 isolated from/by ___________________
DNA Libraries
Notes on cDNA
Genetic Engineering Experiment
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Four stages
– DNA cleavage
 ______________________ cleaves source DNA into fragments
– production of ______________________
 DNA fragments inserted into _____________________
– Cloning
– Screening
 clones with ___________________________ identified from clone library
 preliminary screening - eliminate any clones ________________________
_____________________________________
• employ vector with gene for _____________________ and lac Z’ gene
• expose to growth medium
Secondary screening (gene of interest)
– hybridization - cloned genes form base pairs with ____________________________
___________________________________
 grow on agar then ___________________________________
 treat filter with ___________________________ and perform autoradiography
Genome Maps
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Genetic maps show the ______________________________ on a chromosome as
determined by ___________________________________.
– measured in centimorgans (cM)
 one cM = 0.01% recombination
Physical maps are diagrams showing the __________________________ within specific DNA
sequences.
– measured in base-pairs (bp)
– 1,000 base pairs equal 1 kilobase (kb)
 use _________________________ to cut sequences
 use sequenced-tagged sites (STSs) to construct large genome maps
Physical Maps with Restriction Enzymes
Physical Maps with Sequence-Tagged Sites
Genome Sequencing
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Sequencing
– sequencing of entire genomes now practical due to technological advances
 sequencers provide accurate sequences for DNA segments up to ________ long
 five to ten genome copies sequenced to reduce _______________
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Artificial chromosomes
– vector used in cloning _________________ of DNA Larger than what??_________
 ______________________________ (YAC)
 ______________________________ (BAC)
Sequencing by whole genomes
– clone-by-clone sequencing - cloning larger inserts in BAC requires construction of a
physical map, then placing site of BAC clones for later sequencing
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