Biol3451 Ch19 Lect

Recombinant DNA
Technology
Chapter 19
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
Cloning DNA Molecules
• Purify DNA from desired source
• Cut DNA with restriction endonuclease
• Join fragments to cloning vectors cleaved with
compatible restriction endonuclease to create
recombinant DNA molecules
• Transfer recombinant molecules to host cells
• Isolate DNA from individual clones of
transformed host cells
• Do as you please with the isolated DNA
segments…
Restriction Endonucleases
• Hundreds now identified
• Host cell defense against invading DNAs
• Cleave DNA at or near a specific recognition
sequence
– Creates “restriction fragments”
– Recognition sites can be from 4 to 8+ base pairs and
are commonly palindromes
• First one isolated was EcoRI from E. coli
• Often produce “sticky ends”
Restriction
Endonucleases
DNA Cleavage By EcoRI
Recombinant DNA Molecules
Cloning Vectors
• Plasmids
• Phage
• Cosmids
• BACs
• YACs
Plasmids
• Circular independent
replicons, origin of
replication (ori)
• Generally encode useful
but not essential genes
– E.g. antibiotic resistance or
catabolic pathways
• Allow cloning fragments
up to about 10 kbp
• Selectable markers
• Multiple cloning sites
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
• Plasmid has portion of lacZ
gene flanking MCS lacZ
• Host strain has lacZ gene Complementation
missing this seqeunce
• Expression of two
components still yields a
functional LacZ
– Like it has 2 subunits
• Cloning into MCS
eliminates
complementation
• X-gal in media allows for
blue/white selection
Bacteriophage Vectors
• Commonly based upon
phage
• Most internal genes
deleted
• Insert DNA into middle
region (up to10-15 kb)
• Package
• Infect host cells with
integrated helper phage
to provide missing
protein-encoding genes
Cosmids
• Plasmid with phage
packaging sequence
(cos)
• Can clone up to 50 kb
• Packaged into  particles
and injected into host
cells
• Circularizes in cell and
continues as a large
plasmid
BACs
• Bacterial artificial
chromosomes
• Can clone up to 200+ kb
DNA fragments
• Based upon F plasmid
• Origin, selectable marker,
promoters to expressed
cloned genes
YACs
• Yeast artificial
chromosomes
• Have centromere,
telomeres and an
origin of replication,
plus selectable
markers
• Cloned segments of
250 kb
Expression
Vectors
• Also include
regulatable high level
expression promoter
– T7 phage promoter
– lac operator
– lac repressor gene
First Prokaryotic Host Cells
• First clones introduced into strains of E. 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
Cloning into Plasmids
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
Eukaryotic Expression Vectors
• Generally similar to prokaryotic ones
– Commonly start with prokaryotic vector
• Construction done in E. coli
• Shuttle vector
– Origin
– MCS
– Selectable marker
– High expression regulatable promoter
– But also generally an intron included
• Especially for cDNA clones
Cloning into Plant Cells
• Vectors based upon

Ti plasmid
– Derived from
Agrobacterium
tumifaciens plasmid
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???
Transgenic Mammals
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 molecular
biology/genetics/forensics/and everything
– Day earth changed…
PCR Procedure
• Denature DNA
• Add primers, thermostabile
DNAP, dNTPs
• Extension
• Denaturation
• Bind primers
• Extension
• Repeat last 3 steps thirty times
Chromosome
Sorting
• Important for early portion
of human genome project
– Simplified sequencing effort
– Fluorescent tags on specific
chromosomes
Pulse Field Gel Electrophoresis
• For very large DNA

molecules
– To left are intact yeast
chromosomes
• Electric field is
pulsed/changed after
very short intervals
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
cDNA Synthesis
• Oligo T primer
• Reverse transcriptase and
deoxyribonucleotides
• RNaseH
• DNAP I makes second
strand using the hairpin
created by reverse
transcriptase as a primer
• Hairpin cleave by S1
nuclease
Library Screening
• General approach used for
both genomic and cDNA
libraries
– DNA from colonies/plaques
– transferred to membrane
– Denature
– Hybridize with probe
– Detect probe binding
• Method depends upon
labeling of probe
– Isolate specific probe-
binding clones and
culture
Restriction
Maps
• Useful but now
most commonly
generated by
computer from
actual DNA
sequence
Southern
Transfer
• Developed by E. M.
Southern
• Method for
transferring DNA
from a gel to a
membrane
• Described to right
• Also Western and
Northern blots
– Proteins and RNA
Hybridization
• Binding of a probe (generally RNA or DNA) to a

nucleic acid in a gel or more commonly bound to a
membrane
DNA
Sequencing
• Sanger
• Method
described
to right
Chain Termination DNA
Sequencing
• Autoradiograph of results
from Sanger
dideoxyribonucleotide chain
termination method
• Sequence is read from bottom
to top
Fluorescent
Dyes
• DNA sequencing with
fluorescent dyes
attached to chain
terminator
dideoxyribonucleotides
• Allows for automated
DNA sequencing
Electropherogram

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Recombinant DNA

• Expression is sometimes desirable in eukaryotic cells

• Vectors based upon

• Especially for model systems

• For very large DNA

• Binding of a probe (generally RNA or DNA) to a

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