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Biol3451 Ch19 Lect

Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

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44 views37 pages

Biol3451 Ch19 Lect

Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

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maniiiiiiii
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© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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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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