biol3451 ch19 lect
TRANSCRIPT
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Recombinant DNATechnology
Chapter 19
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Recombinant DNA Technology 1971 paper by Kathleen Dana and Daniel
Nathans described isolation of enzyme that
cleaved DNA at specific sequences
1978 Nobel Prize to Nathans, Smith and
Arber for restriction endonuclease
discovery
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Cloning DNA Molecules
Purify DNA from desired source
Cut DNA with restriction endonuclease
Join fragments to cloning vectors cleaved with
compatible restriction endonuclease to createrecombinant DNA molecules
Transfer recombinant molecules to host cells
Isolate DNA from individual clones oftransformed host cells
Do as you please with the isolated DNAsegments
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Restriction Endonucleases
Hundreds now identified
Host cell defense against invading DNAs
Cleave DNA at or near a specific recognitionsequence
Creates restriction fragments
Recognition sites can be from 4 to 8+ base pairs and
are commonly palindromes
First one isolated wasEcoRI fromE. coli
Often produce sticky ends
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Restriction
Endonucleases
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DNA Cleavage ByEcoRI
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Recombinant DNA Molecules
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Cloning Vectors Plasmids
Phage
Cosmids
BACs
YACs
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Plasmids
Circular independent
replicons, origin of
replication (ori)
Generally encode usefulbut not essential genes
E.g. antibiotic resistance or
catabolic pathways
Allow cloning fragmentsup to about 10 kbp
Selectable markers
Multiple cloning sites
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Plasmid Cloning Vectors
As small as possible
with minimum
restriction endonuclease
cutting sites in genes
ori
Selectable marker(s)
Multiple cloning site(MCS)
Reporter function useful
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Cosmids
Plasmid with Pphage
packaging sequence
(cos)
Can clone up to 50 kb Packaged into Pparticles
and injected into host
cells
Circularizes in cell andcontinues as a large
plasmid
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BACs
Bacterial artificial
chromosomes
Can clone up to 200+ kbDNA fragments
Based upon F plasmid
Origin, selectable marker,
promoters to expressed
cloned genes
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YACs
Yeast artificial
chromosomes
Have centromere,telomeres and an
origin of replication,
plus selectable
markers
Cloned segments of
250 kb
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Expression
Vectors Also include
regulatable high level
expression promoter T7 phage promoter
lac operator
lac repressor gene
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First Prokaryotic Host Cells
First clones introduced into strains ofE. coli K12
Protocol
Isolate target DNA
Cut with RE
Ligate to vector
Transform host cells
Plate on antibiotic-containing medium
Identify recombinant plasmids
Identify/characterize specific clones
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Cloning into Plasmids
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Expression of Recombinant
Genes in Eukaryotes
Expression is sometimes desirable in eukaryotic cells
Especially if post-translational modifications are important or
study simply requires it
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Eukaryotic Expression Vectors
Generally similar to prokaryotic ones
Commonly start with prokaryotic vector
Construction done inE. coli
Shuttle vector
Origin
MCS
Selectable marker High expression regulatable promoter
But also generally an intron included
Especially for cDNA clones
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Cloning into Plant Cells
Vectors based upon
Ti plasmid
Derived fromAgrobacterium
tumifaciensplasmid
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Cloning into Mammalian Hosts
Especially for model systems
DNA integrates into a host cell
chromosome
Random vs. site-directed
Transgenics
Models for diseases
Improved individuals???
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Transgenic Mammals
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Polymerase Chain Reaction
PCR
1993Nobel Prize for Kary Mullis
Proposed in 1986 Can provide millions of fold amplification of
a DNA fragment or sequence
Needle in haystack
Revolutionized molecularbiology/genetics/forensics/and everything
Day earth changed
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PCR Procedure
Denature DNA
Add primers, thermostabile
DNAP, dNTPs
Extension
Denaturation
Bind primers
Extension Repeat last 3 steps thirty times
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Chromosome
Sorting Important for early portion
of human genome project
Simplified sequencing effort Fluorescent tags on specific
chromosomes
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Pulse Field Gel Electrophoresis
For very large DNA
molecules
To left are intact yeastchromosomes
Electric field is
pulsed/changed after
very short intervals
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cDNAs Copies of mRNAs in DNA
By reverse transcriptase
Needed to analyze genes and also to express
eukaryotic genes in prokaryotic systems
Introns
cDNA libraries
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cDNA Synthesis
Oligo T primer
Reverse transcriptase and
deoxyribonucleotides
RNaseH
DNAP I makes second
strand using the hairpin
created by reversetranscriptase as a primer
Hairpin cleave by S1
nuclease
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Library Screening
General approach used forboth genomic and cDNAlibraries
DNA from colonies/plaques
transferred to membrane Denature
Hybridize with probe
Detect probe binding
Method depends uponlabeling of probe
Isolate specific probe-binding clones and
culture
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Restriction
Maps
Useful but now
most commonlygenerated by
computer from
actual DNA
sequence
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Southern
Transfer Developed by E. M.Southern
Method for
transferring DNAfrom a gel to a
membrane
Described to right
Also Western and
Northern blots
Proteins and RNA
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Hybridization
Binding of a probe (generally RNA or DNA) to a
nucleic acid in a gel or more commonly bound to amembrane
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DNA
Sequencing
Sanger
Method
describedto right
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Chain Termination DNA
Sequencing
Autoradiograph of results
from Sanger
dideoxyribonucleotide chaintermination method
Sequence is read from bottom
to top
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Fluorescent
Dyes
DNA sequencing with
fluorescent dyes
attached to chain
terminator
dideoxyribonucleotides
Allows for automatedDNA sequencing
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Electropherogram