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Biotechnologies
DNA Technologies Overview
Natalia Volodina, Ph.D.
CHAPTER 9
DNA Technologies
Key topics:
– DNA cloning techniques
– DNA analysis methods
– DNA microarray technology
– Expression of recombinant proteins
Recombinant DNA
• Artificially created DNA that combines sequences
that do not occur together in the nature
• Basis of much of the modern molecular biology
– Molecular cloning of genes
– Over-expression of proteins
– Transgenic food, animals …
DNA Cloning
• Organism cloning:
– Creation of identical copies of an organism
• DNA cloning:
– Creation of identical copies of a piece of DNA (gene)
– isolate a specific gene from the source organism and amplify it in
the target organism
• Basic steps:
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Cut the source DNA at the boundaries of the gene
Select a suitable carrier DNA (vector)
Insert the gene into the vector
Insert the recombinant vector into host cell
Let the host produce multiple copies of recombinant DNA
DNA Cloning: General Scheme
Restriction Endonucleases
• Cleave DNA phosphodiester bonds at specific
sequences
• Common in bacteria
– Eliminates infectious viral DNA
• Some make staggered cuts
– Sticky ends
• Some make straight cuts
– Blunt ends
• Large number are known:
– Commercially available
– Well documented: REBASE
– http://rebase.neb.com/rebase/rebase.html
Cloning Vectors
• Plasmids
– Circular DNA molecules that are separate from the
bacterial genomic DNA
– Can replicate autonomously
– Carry antibiotic resistance genes
– Allows cloning of DNA up to 15,000 bp
• Bacteriophage l
– Virus that infects bacteria
– Efficient delivery of DNA
– Allows cloning of DNA up to 23,000 bp
DNA Ligase
• Enzyme that covalently joins two DNA fragments
– Normally function in DNA repair
– Human DNA ligase uses ATP
– Bacterial DNA ligase uses NAD
Antibiotic Selection
• Antibiotics, such as penicillin and ampicillin, kill
bacteria
• Plasmids can carry genes that give host
bacterium a resistance against antibiotics
• Allows growth (selection) of bacteria that have
taken up the plasmid
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Identification of Empty Plasmids
Colony Lifts
• Bacterial colonies can grow on agar plates
• Each colony is made of descendants of a single
bacterium—cells within a colony are clones
• Colonies may differ from each other
• Colonies can be lifted from the agar plate to the
nitrocellulose paper
• Lifted cells can be broken up by alkali treatment
Detection of Specific DNA
Sequences
• Upon cell lysis, DNA (and cellular proteins) stick to
the paper
• Specific probes can be used to detect the presence
of specific macromolecules
• To detect specific DNA, a radiolabeled DNA probe,
complementary to the target sequence, is used.
• How would you detect specific proteins?
Polymerase Chain Reaction: Steps
• Allows to amplify DNA in the test tube
– Can amplify regions of interests (genes) within a linear DNA
– Can amplify complete circular plasmids
• Mix together
– Target DNA
– Primers (oligos complementary to target)
– Nucleotides: dATP, dCTP, dGTP, dTTP
– Thermostable DNA polymerase
• Place the mixture into thermocycler
– Melt DNA at about 95 ºC
– Cool separated strands to about 50-60 ºC
– Primers anneal to the target
– Polymerase extends primers in 5’3’ direction
– After a round of elongation is done, repeat steps
Outline of PCR
Site-Directed Mutagenesis
• Understanding the function of proteins often
requires that a specific amino acid residue be
mutated
• To mutate an amino acid, change the
nucleotide(s) in the coding DNA and express the
mutated gene
• Site-directed mutagenesis usually relies on
chemically synthesized mutated primers that are
incorporated into newly synthesized DNA
Separation of DNA by Electrophoresis
• Negatively charged DNA migrates to the anode in
the presence of an electric field
• Agarose gel hinders the mobility of DNA molecules
• Mobility depends on the size and the shape
– Small molecules faster
– Compact molecules faster
• Practical use is
– DNA analysis
– DNA purification
– DNA-protein interaction studies
DNA Sequence Analysis
• Sequencing
– Chain termination method (Sanger
sequencing)
– Sequencing by synthesis (Pyrosequencing)
• Single Nucleotide Polymorphism
– Molecular beacons
Genomic Sequencing
DNA Fingerprinting
• Each individual has a unique DNA sequence
• Differences cause variations in the length of fragments
that form when sample is treated with restriction enzymes
– RFLP stands for restriction fragment length polymorphism
• Fragments can be separated by size using agarose gel
electophoresis
• Separated DNA fragments can be blotted on the
membrane (Southern blot)
• Few of the blotted DNA fragments are visualized by
hybridizing with a labeled nucleic acid probe
• Allows matching “suspect” samples to known individuals
The Southern Blot Analysis of RFLP
Expression of Cloned Genes
• We want to study the protein product of the gene
• Special plasmids, called expression vectors,
contain sequences that allow transcription of the
inserted gene
• Expression vectors differ from cloning vectors by
having
– Promoter sequences
– Operator sequences
– Code for ribosome binding site
– Transcription termination sequences
Purification of Recombinant
Genes
• Purification of natural proteins is difficult
• Recombinant proteins can be tagged for
purification
• The tag binds to the affinity resin and thus
captures the fusion protein
– GST
– His-Tag
Eukaryotic Gene Expression in
Bacteria
• An eukaryotic gene from the eukaryotic genome will
not express correctly in the bacterium
• Eukaryotic genes have
– Exons: coding regions
– Introns: noncoding regions
• Introns in eukaryouric gene pose problems
• Bacteria cannot splice introns out
• mRNA is intron-free genetic material
Construction of cDNA
• mRNA can be extracted from eukaryotic cells
• All mRNA molecules have
poly-A tail
– helps in purification of mRNA
– serves as an universal template
• DNA strand can be synthesized using mRNA as a
template
• This is catalyzed by the reverse transcriptase
• The end result is a hybrid where the DNA strand is
complementary to the mRNA
• The hybrid can be converted to duplex DNA,
known as cDNA
DNA Microarrays: Applications
DNA Microarrays allow simultaneous screening
of many thousands of genes: high-throughput
screening
• genome wide genotyping
– Which genes are present in this
individual?
• tissue-specific gene expression
– Which genes are used to make
proteins?
• mutational analysis
– Which genes have been mutated?
DNA Microarrays: Design
Two fundamental approaches
• One-color array
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Patented and commerialized by Affymetrix
Photolitographic synthesis of probe DNA on the chip
Targets are biotin labeled
Bound targets detected using streptavidin-fluorofore
complex
Widely used in industry
Two-color array
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Developed by Stanford University, 1996
Probes sometimes pipetted on the chip
Targets linked to either green or red fluorescent labels
Used often in academia
One-color Quantitative
Microarray Technology
• General Process
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Target is biotin-labeled cRNA
Probe is single-stranded DNA oligo attached to the wafer
Complementary target and probe hybridize
Duplex stained with the streptavidin-bound fluorescent marker
• In situ oligonucleotide synthesis (Affymetrix U.S. patent 5,861,242 )
– 5-inch square quartz wafer with covalently bound layer of silane
– Parallel synthesis with photolithographic masks
– Makes oligos 20-25 nucleotides long; 400,000 per chip
Photolitographic Synthesis of DNA
GeneChip®: Detection
• Bound nucleic acid duplexes have a covalently attached biotin
• Biotin binds a streptavidin-phycoerythrin complex
• Phycoerythrin is a dye from red algae
– Absorbs blue light at 488 nm
– Gives off fluorescent light at about 600 nm
• Fluorescence intensity is proportional to concentration of bound
nucleic acid duplexes
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Phycoerythrin (pigment in red alga)
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Two-Color DNA Microarray
“Genome Chips”
Fluorescent Spots in Two-color
DNA Microarray
Chapter Summary
In this chapter, we learned how:
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to make recombinant DNA
to use bacteria for DNA cloning
to analyze DNA by size and sequence
to mutate and amplify DNA in test-tube
to express and purify eukaryotic genes
to determine expression levels of genes