Isolation and Purification of Nucleic Acids: Sample Processing

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Transcript Isolation and Purification of Nucleic Acids: Sample Processing

Pan, Cerberus, and the Centaurs: Genetic
Engineering
Donna C. Sullivan, PhD
Division of Infectious Diseases
University of Mississippi Medical
Center
Fun Things To Do With DNA
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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
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Prokaryotic DNA
Eukaryotic DNA
Viral DNA or RNA
Plasmid DNA
Isolating Nucleic Acids for
Molecular Analysis
Cell Lysis
Enzymatic or
Chemical
Extraction
General DNA Isolation Procedures
Lyse Cells
• Detergent dissolves cell membrane and denatures protein.
• EDTA chelates divalent cations required by nucleases.
• Proteinase K degrades proteins.
Organic Extraction Nonorganic Extraction Solid Phase Extraction
• Uses
phenol:chloroform:isoamyl
alcohol
• Denatures and removes
proteins
•Toxic Chemicals!!
•Salt precipitation of protein
• DNA precipitation
•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
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DNA (or RNA) is characterized using several
different methods for assessing quantity,
quality, and molecular size.
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UV spectrophotometry
Agarose gel electrophoresis
Colorimetric blotting
How Much DNA Do You Have?
Absorbance from UV Spectrophotometry
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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
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Concentration of RNA =
(A260-A320) X dilution factor X 40 µg/mL
How Does Your DNA Look? Quality
from Agarose Gel Electrophoresis
Human Whole Blood DNA
Lambda DNA
marker
Whole blood genomic DNA
Lambda DNA cut with
Hind III marker
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 Analysis
of Nucleic Acids
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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
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Restriction
endonucleases
DNA polymerases
(synthesize DNA)
DNA ligases (join
DNA strands)
Kinases
(phosphorylation of 5´ends of DNA)
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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
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>2000 distinct restriction enzymes have been
identified
Recognize symmetrical dsDNA (palindromes)
 Utilized in the digestion of DNA molecules
 Nomenclature:
EcoRI
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First letter of Genus + first 2 letters of species + order of enzyme discovery
E
co
RI
Restriction Enzymes Recognize
Palindromes
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Palindrome reads the same in both directions
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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
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
5’GATATC3’
3’CTATAG5’
5’GAATTC3’
3’CTTAAG5’
1
2
PstI
5’CTGCAG3’
3’GACGTC5’
3
Microcoleus
MstII
5’CCTNAGG3’
3’GGANTCC5’
2,4
Nocardia ototidis caviarum
NotI
5’GCGGCCGC3’
3’CGCCGGCG5’
2,6
Haemophilus aegyptius
EcoRI
Providencia stuartii
1.
2.
3.
Enzyme produces blunt ends..
Single strand is 5’ strand.
Single strand is 3’ strand.
4.
5.
6.
N= any purine or pyrimidine pair.
Enzyme cuts 8 nts 3’ of recognition site.
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.
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Gel Electrophoresis
 Electrophoresis
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
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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.
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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
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1
2
3
4
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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.
Restriction Enzyme Mapping
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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
BamH1
XhoI
BamH1
4.3 kb
3.7 kb
2.8 kb
4.0 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
1.1 kb
2.8 kb
1.7 kb
1.7 kb
XhoI
1.2 kb 1.2 kb
1.2 kb
1.1 kb
0.7 kb
XhoI
Mapping DNA: Southern Blots
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DNA immobilized on solid support
Detect specific DNA fragments with a DNA
probe using hybridization
Ok, what the #&*^!!! is a probe?
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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
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To clone
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produce multiple identical copies of something
A clone
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identical copy, derived from single progenitor
may be DNA molecules, cells, or an organism
Molecular Cloning
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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 Plasmid
Replicator (ori)
Selectable marker
Cloning site
Cloning into Plasmid Vectors
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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
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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
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Treat with CaCl2
Add media with
antibiotic, incubate
Streak on selective
media plate
High copy number
plasmids give the best
yield
Efficiency Of Transformation
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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
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Antibiotic selection
Color selection
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 galactosidase gene (Lac Z)
Green fluorescent protein (GFP)
Selection Of Clones Containing Inserts
Intact lac Z gene
beta gal= blue colonies
Interrupted lac Z gene
No beta gal= white colonies
Bacteriophage Vectors
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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
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Accommodate large inserts
Extremely stable
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Integrate into yeast chromosome
Mini-chromosomes
Artificial chromosome
Eukaryotic system
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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?
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Provide large quantities
of DNA for analysis
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Mapping, sequencing
studies
Identification, disease
diagnosis
Provide source of
specific gene product
for commercial use
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Production of medically
important molecules
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Provide source of
specific genes for
creation of transgenic
animals
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IS THERE A BETTER
WAY TO DO SOME
OF THIS?
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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)
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Cary Mullis and PCR
Wanted a way to generate large
amounts of DNA from a single
copy
 Initially used the “3 graduate
student” method
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Denaturing (unwinding) DNA
 Annealing (hybridizing) primers
 Extending (copying) DNA
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THREE STEPS OF PCR
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Denaturation of target (template)
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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”
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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.
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Thermostable DNA Polymerase: Yellowstone
National Park And Deep Sea Vents
Thermostable Polymerases
Polymerase
Taq pol
T ½,
95oC
40 min
Extension Type of
Rate (nt/sec)
ends
75
3’A
Source
T. aquaticus
Amplitaq
(Stoffel
fragment)
Vent
80 min
>50
3’A
T. aquaticus
400 min
>80
Deep Vent
1380 min
?
Pfu
>120 min
60
95%
blunt
95%
blunt
Blunt
Tth
20 min
>33
3’A
Thermococcus
litoralis
Pyrococcus
GB-D
Pyrococcus
furiosus
T.
thermophilus
Taq: Thermus aquaticus (most commonly used)
PCR Cycle: Temperatures
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Denaturation temperature
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Annealing temperature
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Reduce double stranded molecules to single
stranded molecules
Controls specificity of hybridization
Extension temperature
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Optimized for individual polymerases
Combinations Of Cycle Temperatures
TEMP
94-60-72
94-55-72
94-50-72
94-48-68
94-45-65
94-37-65
FOR
COMMENTS
Perfect, long
primers
Good or perfectly
matched primers
between 19-24 nt
Adequate primers
Higher temp can be used;
maximum annealling temp
Standard conditions
Poorly matched
primers
Unknown match,
likely poor
Hail Mary
Allows 4-5 mismatches/20 nt
Allows 1-3 mismatches/20 nt
Primers of questionable
quality, long-shot PCR
Uncontrolled results
REAL TIME PCR
Detects PCR products as they accumulate
 Detect ds DNA by two methods:
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Intercalator fluorescent markers (ethidium
bromide, syber green dye): non specific
 Fluorogenic probes: specific
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Plot increase in fluorescence versus cycle
number
GEL ANALYSIS VS
FLUORESCENCE
DNA Detection: SYBR Green I Dye
DENATURATION STEP: DNA + PRIMERS + DYE
WEAK BACKGROUND FLUORESCENCE
ANEALING STEP:DYE
BINDS dsDNA, EMITS LIGHT
EXTENSION STEP: MEASURE
LIGHT EMMISSION
Cycle Threshold (Ct)
Cycle
Threshold
Threshold line
(fluorescent
units)
Construction of Standard Curve
Threshold fluorescence
level
Threshold cycles for each sample
Real-Time PCR Labeled Probes
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Cleavage-based probes
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Molecular beacons
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TaqMan Assay
Fluorescent reporter at 5’ end and a quencher at 3’ end
Hairpin loop structure
Fluorescent reporter at 5’ end and a quencher at 3’ end
FRET probes
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Fluorescence resonance energy transfer probes
What is DNA Sequencing?
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DNA sequencing is the ability to determine
nucleotide sequences of DNA molecules.
DNA Sequencing Methods
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Technology
Chain termination
 Cycle sequencing
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Chemistry
Maxam and Gilbert
 Sanger
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Platform
Manual
 Automated
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Sanger (Dideoxy) DNA Sequencing
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Incorporation of 2´,3´-dideoxynucleotides by
DNA polymerase
Termination of elongation reaction
Fragment size analysis (manual vs.
automated)
5 CH OH
OH
2
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Gel
Capillary
O
4
C1
C
H
H
3C
C
2,3-dideoxyribose H
H
2
Dideoxy or Sanger DNA Sequencing
A
T
G
C
A
ATTA
ATTAGA
AT
ATT
ATTAGACGT
ATTAG
ATTAGACG
ATTAGAC
A T
ATTAGACGT
G C
Sequencing Gels
Cycle Sequencing
Cycle sequencing is chain termination
sequencing performed in a thermal cycler.
 Cycle sequencing requires a heat-stable DNA
polymerase.
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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.
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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.
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A
T
G
T
AC
GT
The fragments are
distinguished by size and
“color.”
Dye Terminator Sequencing
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The DNA ladder is resolved in one gel lane
or in a capillary.
G
A
T
GA
TC
C
G
T
C
T
G
A
Slab gel
Capillary
Dye Terminator Sequencing
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The DNA ladder is read on an
electropherogram.
Slab gel
Capillary
Electropherogram
5′ AGTCTG
Automated Sequencing
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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.
T/T
5′ AGTCTG
T/A
5′ AG(T/A)CTG
A/A
5′ AGACTG
APPLICATIONS OF GENETIC ENGINEERING
in
Produce
Transgenics
Medicine
May be
includes
Plant
Genetic
screening
Gene
mapping
Gene
therapy
Raise
Legal and
ethical questions
Forensic
medicine
To
affect
Yield
Disease resistant
Herbicide
resistance
Drought, salt and
cold tolerance
Storage,
appearance
Raise
or
Animal
to
Increase
yield
For?
Organ
donors
and
Production
therapeutic
proteins