Pan, Cerberus, and the Centaurs: Genetic Engineering
Donna C. Sullivan, PhDDivision of Infectious DiseasesUniversity of Mississippi Medical Center
Fun Things To Do With DNA Spool it onto a glass rod (Isolation) Put it in a necklace (Precipitation) Pull it apart (Denature) and put it back together (Anneal) Cut it up (Restriction Enzymes) and look at it (Gel
electrophoresis) Map it (Southern Blots) Read it (Sequencing) Copy it millions of times (Cloning, Polymerase Chain
Reaction) Move it from one place to another (Genetic Engineering)
Diversity of Genetic Material Prokaryotic DNA Eukaryotic DNA Viral DNA or RNA Plasmid DNA
Enzymatic or ChemicalExtraction
Cell Lysis
Isolating Nucleic Acids forMolecular Analysis
General DNA Isolation ProceduresLyse Cells
• Detergent dissolves cell membrane and denatures protein.• EDTA chelates divalent cations required by nucleases.
• Proteinase K degrades proteins.
Organic Extraction• Uses
phenol:chloroform:isoamyl alcohol
• Denatures and removes proteins
•Toxic Chemicals!!
Nonorganic Extraction•Salt precipitation of protein
• DNA precipitation
Solid Phase Extraction• Apply sample
• Wash• Elute DNA
• Best method!
(1 X 107 cells) X (6 pg DNA/cell) X 80% yield= 48 mg DNA!!!
Looking at DNA: Nucleic Acid Analysis DNA (or RNA) is characterized using several
different methods for assessing quantity, quality, and molecular size. UV spectrophotometry Agarose gel electrophoresis Colorimetric blotting
How Much DNA Do You Have? Absorbance from UV Spectrophotometry DNA and RNA absorb maximally at a
wavelength of 260 nm. Proteins absorb at 280 nm. Background scatter absorbs at 320 nm. Concentration of DNA =
(A260 – A320) X dilution factor X 50 µg/mL Concentration of RNA =
(A260-A320) X dilution factor X 40 µg/mL
Lambda DNA marker
Human Whole Blood DNA
Lambda DNA cut with Hind III marker
Whole blood genomic DNA
How Does Your DNA Look? Quality from Agarose Gel Electrophoresis
Pulling DNA Apart And Putting It Back Together: Denaturation and Annealing Reactions
DNA Likes To Find Its Perfect Match: Denaturation and Annealing of DNA
Hybridization Will Occur In Liquid Or On A Solid Surface
Melting Temperature (Tm), Salt and G + C Content
Basic Techniques for Analysisof Nucleic Acids Endonuclease digestion (DNAse, RNase,
restriction enzymes) Electrophoresis (agarose and polyacrylamide
gel electrophoresis) Enzymatic modification (polymerase, kinase,
phosphatase, ligase)
Cutting, Chewing, Tagging DNA: Nucleic Acid Modifying Enzymes Restriction endonucleases DNA polymerases (synthesize DNA) DNA ligases (join DNA strands) Kinases (phosphorylation of 5´-ends of DNA)
Phosphatases (dephosphorylate 5´-ends of DNA)
Ribonucleases (digest RNA molecule. Example: RNase A)
Deoxyribonucleases (digest DNA molecules)
Restriction Endonucleases (RE) Found only in microorganisms Exhibit novel DNA sequence specificities
>2000 distinct restriction enzymes have been identified
Recognize symmetrical dsDNA (palindromes) Utilized in the digestion of DNA molecules Nomenclature:
EcoRI
First letter of Genus + first 2 letters of species + order of enzyme discovery E co RI
Restriction Enzymes Recognize Palindromes Palindrome reads the same in both directions
BOB “Able was I ere I saw Elba.” (Napoleon
Bonapart, following his exile from the European continent to the island of Elba)
Sequences directly opposite one another on opposite strands of the ds DNA molecule
MICROORGANISM ENZYME SEQUENCE NOTES
Haemophilus aegyptius HaeIII 5’GGCC3’3’CCGG5’
1
Thermus aquaticus TaqI 5’TCGA3’3’AGCT5’
2
Haemophilus haemolyticus HhaI 5’GCGC3’3’CGCG5’
3
Desulfovibrio desulfuricans DdeI 5’CTNAG3’3’GANTC5’
2,4
Moraxella bovis MboII 5’GAAGA(N)83’3’CTTCT(N)75’
4,5
Escherichia coli EcoRV
EcoRI
5’GATATC3’3’CTATAG5’5’GAATTC3’3’CTTAAG5’
12
Providencia stuartii PstI 5’CTGCAG3’3’GACGTC5’
3
Microcoleus MstII 5’CCTNAGG3’3’GGANTCC5’
2,4
Nocardia ototidis caviarum NotI 5’GCGGCCGC3’3’CGCCGGCG5’
2,6
1. Enzyme produces blunt ends..2. Single strand is 5’ strand.3. Single strand is 3’ strand.
4. N= any purine or pyrimidine pair.5. Enzyme cuts 8 nts 3’ of recognition site.6. Cuts mammalian DNA very rarely.
Looking At DNA: Electrophoresis Nucleic acids are separated based on size and
charge. DNA molecules migrate in an electrical field Employs a sieve-like matrix (THINK JELLO!)
and an electrical field. DNA is negatively charged and migrates
towards the positively charged anode.
Gel ElectrophoresisElectrophoresis is the movement of molecules
by an electric current.
Nucleic acid moves from a negative to a positive pole.
Nucleic acid has a net negative charge, they RUN TO RED
Principles of Gel Electrophoresis The gel itself is composed of either agarose or
polyacrylamide Agarose is a polysaccharide extracted from
seaweed Polyacrylamide is a cross-linked polymer of
acrylamide. Acrylamide is a potent neurotoxin and should be
handled with care!
“Submarine” Agarose Gel Electrophoresis
Agarose Gel Apparatus
Comparison Of Various Agarose Concentrations
Electrophoresis Of Lambda DNA Digested Using Three Different RE
Lane 1 contains uncut lambda DNA.
Lane 2 contains lambda DNA digested by PstI.
Lane 3 contains lambda DNA digested by EcoRI.
Lane 4 contains lambda DNA digested by HindIII.
1 2 3 4
Restriction Enzyme Mapping Digest DNA with a restriction enzyme. Resolve the fragments by gel electrophoresis. The number of bands indicates the number of
restriction sites. The size of the bands indicates the distance
between restriction sites.
Restriction Enzyme Mapping: Circular DNA Molecules
BamH1
XhoI
XhoI
1.1 kb
1.7 kb
1.2 kb
2.8 kb4.3 kb3.7 kb
2.3 kb1.9 kb
1.4 kb1.3 kb
0.7 kb
BamH1 XhoIBamH1XhoI
4.0 kb
2.8 kb
1.2 kb
1.7 kb
1.2 kb
1.1 kb
Mapping DNA: Southern Blots
DNA immobilized on solid support Detect specific DNA fragments with a DNA
probe using hybridization Ok, what the #&*^!!! is a probe?
It’s a usually a clone or amplified DNA—we’ll get there in a minute.
Southern Blot Hybridization: Overview
Southern Blot Analysis of EHV-3 DNA
Restriction Enzyme Map of EHV-3 DNA
BamHI
BclI
BglII
EcoRI
Hind III
CLONE: THE NOUN AND THE VERB
To clone produce multiple identical copies of something
A clone identical copy, derived from single progenitor may be DNA molecules, cells, or an organism
Molecular Cloning Genetic engineering
includes techniques that allow for the construction of novel DNA molecules by joining DNA sequences from different sources. Recombinant
DNA
Vector
Clone
Cloning PlasmidReplicator (ori)
Selectable marker
Cloning site
Cloning into Plasmid Vectors Cut plasmid, target
DNA with RE Treat plasmid DNA
with alkaline phosphatase
Mix plasmid and target DNAs to allow annealing
Add DNA ligase
Transfection And Transformation: Putting Genes Back Into Cells Calcium phosphate/chloride precipitation
Aggregates of DNA precipitate and are endocytosed
DEAE dextran Anion binding gel that aggregates DNA
Biolistics DNA coated onto gold microprojectiles
Electroporation High voltage shock that makes transient DNA
permeable holes in cell membranes
Transform bacterial cells
Treat with CaCl2
Add media with antibiotic, incubate
Streak on selective media plate
High copy number plasmids give the best yield
Efficiency Of Transformation 10% of cells treated take up DNA 1% of cells become stable transformants Most transfections are transient and must be
forced to maintain the foreign DNA by selection pressure Antibiotic selection Color selection
galactosidase gene (Lac Z) Green fluorescent protein (GFP)
Selection Of Clones Containing Inserts
Intact lac Z gene
Interrupted lac Z gene
beta gal= blue colonies
No beta gal= white colonies
Bacteriophage Vectors Most are derived from
lambda phage Charon 16A vectors were
named after the ferryman of Greek mythology who conveyed the spirits of the dead across the River Styx
Yeast Vectors Accommodate large inserts Extremely stable
Integrate into yeast chromosome Mini-chromosomes Artificial chromosome
Eukaryotic system Post-translational modifications similar to
mammalian systems
Remember Eukaryotic Genes Contain Introns: Reverse Transcription of RNA to cDNA
What about primers?Remember poly A tail?
Primers are long string of Ts!
What Are Cloned Genes Good For Anyway? Provide large quantities of DNA for analysis
Mapping, sequencing studies Identification, disease diagnosis
Provide source of specific gene product for commercial use Production of medically important molecules
Provide source of specific genes for creation of transgenic animals
IS THERE A BETTER WAY TO DO SOME OF THIS? PCR and Cary Mullis
Cary Mullis and the Nobel Prize: The Basics Knew that you could expose template DNA by
boiling ds DNA to produce ss DNA Knew that you could use primers to initiate
DNA synthesis Knew that a cheap, commercial enzyme was
available (Klenow fragment of E. coli DNA polymerase)
Cary Mullis and PCR Wanted a way to generate large
amounts of DNA from a single copy
Initially used the “3 graduate student” method Denaturing (unwinding) DNA Annealing (hybridizing) primers Extending (copying) DNA
THREE STEPS OF PCR Denaturation of target (template)
Usually 95oC Annealing of primers
Temperature of annealing is dependent on the G+C content
May be high (no mismatch allowed) or low (allows some mismatch) “stringency”
Extension (synthesis) of new strand
Automation of PCR PCR requires repeated temperature changes. The thermal cycler changes temperatures in a
block or chamber holding the samples. Thermostable (heat stable) polymerases are
used to withstand the repeated high denaturation temperatures.
Thermostable DNA Polymerase: Yellowstone National Park And Deep Sea Vents
Thermostable Polymerases Polymerase T ½,
95oC Extension
Rate (nt/sec) Type of
ends Source
Taq pol 40 min 75 3’A T. aquaticus
Amplitaq (Stoffel
fragment)
80 min >50 3’A T. aquaticus
Vent 400 min >80 95% blunt
Thermococcus litoralis
Deep Vent 1380 min ? 95% blunt
Pyrococcus GB-D
Pfu >120 min 60 Blunt Pyrococcus furiosus
Tth 20 min >33 3’A T. thermophilus
Taq: Thermus aquaticus (most commonly used)
PCR Cycle: Temperatures Denaturation temperature
Reduce double stranded molecules to single stranded molecules
Annealing temperature Controls specificity of hybridization
Extension temperature Optimized for individual polymerases
Combinations Of Cycle TemperaturesTEMP FOR COMMENTS94-60-72 Perfect, long
primersHigher temp can be used;maximum annealling temp
94-55-72 Good or perfectlymatched primersbetween 19-24 nt
Standard conditions
94-50-72 Adequate primers Allows 1-3 mismatches/20 nt
94-48-68 Poorly matchedprimers
Allows 4-5 mismatches/20 nt
94-45-65 Unknown match,likely poor
Primers of questionablequality, long-shot PCR
94-37-65 Hail Mary Uncontrolled results
REAL TIME PCR Detects PCR products as they accumulate Detect ds DNA by two methods:
Intercalator fluorescent markers (ethidium bromide, syber green dye): non specific
Fluorogenic probes: specific Plot increase in fluorescence versus cycle
number
GEL ANALYSIS VS FLUORESCENCE
DNA Detection: SYBR Green I Dye
DENATURATION STEP: DNA + PRIMERS + DYEWEAK BACKGROUND FLUORESCENCE
ANEALING STEP:DYE BINDS dsDNA, EMITS LIGHT
EXTENSION STEP: MEASURE LIGHT EMMISSION
(fluorescent units)
Cycle Threshold
Threshold line
Cycle Threshold (Ct)
Threshold fluorescence level
Threshold cycles for each sample
Construction of Standard Curve
Real-Time PCR Labeled Probes Cleavage-based probes
TaqMan Assay Fluorescent reporter at 5’ end and a quencher at 3’ end
Molecular beacons Hairpin loop structure Fluorescent reporter at 5’ end and a quencher at 3’ end
FRET probes Fluorescence resonance energy transfer probes
What is DNA Sequencing? DNA sequencing is the ability to determine
nucleotide sequences of DNA molecules.
DNA Sequencing Methods Technology
Chain termination Cycle sequencing
Chemistry Maxam and Gilbert Sanger
Platform Manual Automated
Sanger (Dideoxy) DNA Sequencing Incorporation of 2´,3´-dideoxynucleotides by
DNA polymerase Termination of elongation reaction Fragment size analysis (manual vs.
automated) Gel Capillary
2,3-dideoxyribose H
OC
C
C C
CH2OH OH
H
H
H
1
5
4
32
ATTAGACGT
A
AATTAATTAGA
T
ATATTATTAGACGT
G
ATTAGATTAGACG
C
ATTAGAC
A T G C
Dideoxy or Sanger DNA Sequencing
Sequencing Gels
Cycle Sequencing Cycle sequencing is chain termination
sequencing performed in a thermal cycler. Cycle sequencing requires a heat-stable DNA
polymerase.
Fluorescent Dyes Fluorescent dyes are multi-cyclic molecules
that absorb and emit fluorescent light at specific wavelengths.
Examples are fluorescein and rhodamine derivatives.
For sequencing applications, these molecules can be covalently attached to nucleotides.
Dye Terminator Sequencing A distinct dye or “color” is used for each of
the four ddNTP. Since the terminating nucleotides can be
distinguished by color, all four reactions can be performed in a single tube.
ACGT
The fragments are distinguished by size and “color.”
A
T
G
T
Capillary
GT
C
TG
A
Slab gel
GATC G A T C
Dye Terminator Sequencing The DNA ladder is resolved in one gel lane
or in a capillary.
Dye Terminator Sequencing The DNA ladder is read on an
electropherogram.
CapillarySlab gel
5′ AGTCTG
Electropherogram
5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG
T/T T/A A/A
Automated Sequencing Dye primer or dye terminator sequencing on capillary
instruments. Sequence analysis software provides analyzed sequence in text
and electropherogram form. Peak patterns reflect mutations or sequence changes.
APPLICATIONS OF GENETIC ENGINEERING
in Produce
Medicine Transgenics
includes
May be
Genetic screening
Gene mapping
Gene therapy
Forensic medicine
Plant or Animal
To affect
to
YieldDisease resistant
Herbicide resistance
Drought, salt and cold tolerance
Storage, appearance
Increase yield
For?
Organ donors
and
Production therapeutic
proteins
Legal and ethical questions
Raise
Raise
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