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