DNA and RNA isolation and purification (course readings 10 and 11)

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DNA and RNA isolation and purification (course
readings 10 and 11)

• Genomic DNA preparation overview
• Plasmid DNA preparation
• DNA purification
• Phenol extraction
• Ethanol precipitation
• RNA work

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What do we need DNA for?

• Detect, enumerate, clone genes
• Detect, enumerate species
• Detect/sequence specific DNA regions
• Create new DNA constructs (recombinant DNA)

What about RNA?

• Which genes are being transcribed?
• When/where are genes being transcribed?
• What is the level of transcription?

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DNA purification overview
cell harvest and lysis
cell growth
DNA concentration
DNA purification
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Bacterial genomic DNA prep cell extract

• Lysis
• Detergents
• Organic solvent
• Proteases (lysozyme)
• Heat

cell extract
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Genomic DNA prep removing proteins and RNA
chloroform
Need to mix gently! (to avoid shearing breakage
of the genomic DNA)
Add the enzyme RNase to degrade RNA in the
aqueous layer
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2 ways to concentrate the genomic DNA
70 final conc.
Ethanol precipitation
spooling
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Genomic DNA prep in plants -- how get rid of
carbohydrates?
CTAB Cationic detergent (MC 6.61-6.62)
CH3
CH3
(low ionic conditions)
Br-
N
CH3
C16H33
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Plasmids vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
Non-chromosomal DNA Replication independent of
the chromosome Many copies per cell Easy to
isolate Easy to manipulate
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Plasmid purification alkaline lysis
Alkaline conditions denature DNA Neutralize
genomic DNA cant renature (plasmids CAN because
they never fully separate)
10
DNA purification silica binding
Binding occurs in presence of high salt
concentration, and is disrupted by elution with
water
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DNA purification phenol/chloroform
extraction 11 phenol chloroform or 25241
phenol chloroform isoamyl alcohol Phenol
denatures proteins, precipitates form at
interface between aqueous and organic
layer Chloroform increases density of organic
layer Isoamyl alcohol prevents foaming
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Phenol extraction

• Aqueous volume (at least 200 microliters)
• Add 2 volumes of phenolchloroform, mix well
• Spin in centrifuge, move aqueous phase to a new
  tube
• Repeat steps 2 and 3 until there is no
  precipitate at phase interface
• (extract aqueous layer with 2 volumes of
  chloroform)

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Ethanol precipitation (DNA concentration)

• Ethanol depletes the hydration shell surrounding
  DNA
• Allowing cations to interact with the DNA
  phosphates
• Reducing repulsive forces between DNA strands
• Causing aggregation and precipitation of DNA
• Aqueous volume (example 200 microliters)
• -- add 22 microliters sodium acetate 3M pH 5.2
• -- add 1 microliter of glycogen (gives a visible
  pellet)
• -- add 2 volumes (446 microliters) 100 ethanol
• -- mix well, centrifuge at high speed, decant
  liquid
• -- wash pellet (70 ethanol), dry pellet,
  dissolve in appropriate volume (then determine
  DNA concentration)

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DNA purification overview
cell harvest and lysis
cell growth
DNA concentration
DNA purification
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DNA --------------gt mRNA --------------gt
protein
Lots of information in mRNA When is gene
expressed? What is timing of gene
expression? What is the level of gene
expression? (but what does an mRNA measurement
really say about expression of the protein?)
Isolation of RNA -- Course reading 11
16
RNA in a typical eukaryotic cell 10-5
micrograms RNA 80-85 is ribosomal RNA 15-20 is
small RNA (tRNA, small nuclear RNAs) About 1-5
is mRNA -- variable in size -- but usually
containing 3 polyadenylation
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The problem(s) with RNA RNA is chemically
unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular
transesterification RNA is susceptible to nearly
ubiquitous RNA-degrading enzymes
(RNases) RNases are released upon cell
lysis RNases are present on the skin RNases are
very difficult to inactivate -- disulfide
bridges conferring stability -- no requirement
for divalent cations for activity
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Common sources of RNase and how to avoid them
Contaminated solutions/buffers USE GOOD STERILE
TECHNIQUE TREAT SOLUTIONS WITH DEPC (when
possible) MAKE SMALL BATCHES OF
SOLUTIONS Contaminated equipment USE
RNA-ONLY PIPETS, GLASSWARE, GEL RIGS BAKE
GLASSWARE, 300C, 4 hours USE RNase-free PIPET
TIPS TREAT EQUIPMENT WITH DEPC
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• Top 10 sources of RNAse contamination
• (Ambion Scientific website)
• Ungloved hands
• Tips and tubes
• Water and buffers
• Lab surfaces
• Endogenous cellular RNAses
• RNA samples
• Plasmid preps
• RNA storage (slow action of small amounts of
  RNAse
• Chemical nucleases (Mg, Ca at 80C for 5 )
• Enzyme preparations

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Inhibitors of Rnase DEPC diethylpyrocarbonate
alkylating agent, modifying proteins and nucleic
acids fill glassware with 0.1 DEPC, let stand
overnight at room temp solutions may be treated
with DEPC -- add DEPC to 0.1, then autoclave
(DEPC breaks down to CO2 and ethanol)
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Inhibitors of Rnase Vanadyl ribonucleoside
complexes competitive inhibitors of RNAses, but
need to be removed from the final preparation of
RNA Protein inhibitors of RNAse horseshoe-shaped
, leucine rich protein, found in cytoplasm of
most mammalian tissues must be replenished
following phenol extraction steps
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Making and using mRNA (1)
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Making and using mRNA (2)
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Purifying RNA the key is speed Break the
cells/solubilize components/inactivate RNAses by
the addition of guanidinium thiocyanate (very
powerful denaturant) Extract RNA using
phenol/chloroform (at low pH) Precipitate the
RNA using ethanol/LiCl Store RNA in
DEPC-treated H20 (-80C) in formamide
(deionized) at -20C
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Selective capture of mRNA oligo
dT-cellulose Oligo dT is linked to cellulose
matrix RNA is washed through matrix at high salt
concentration Non-polyadenylated RNAs are washed
through polyA RNA is removed under low-salt
conditions (not all of the non-polyadenylated
RNA gets removed
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• Other methods to capture mRNA
• Poly(U) sepharose chromatography
• Poly(U)-coated paper filters
• Streptavidin beads
• A biotinylated oligo dT is added to
  guanidinium-treated cells, and it anneals to the
  polyA tail of mRNAs
• Biotin/streptavidin interactions permit isolation
  of the mRNA/oligo dT complexes

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How good is the RNA prep? The rRNA should form
2 sharp bands in ethidium bromide-stained gels
(but mRNA will not be visible Use radiolabelled
poly dT in a pilot Northern hybridization--should
get a smear from 0.6 to 5 kb on the blot Use a
known, standard gene probe (e.g. GAPDH in
mammalian cells) in Northern hybridization--there
should be a sharp band with no degradation
products
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In vitro amplification of DNA by PCR

• Theory of PCR
• Components of the PCR reaction
• A few advanced applications of PCR
• Reverse transcription PCR (for RNA measurements)
• Quantitative real-time PCR
• PCR of long DNA fragments
• Inverse PCR
• MOPAC (mixed oligonucleotide priming)

Molecular Cloning, p. 8.1-8.24
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What is PCR?

• Polymerase Chain Reaction--first described in
  1971 by Kleppe and Khorana, re-described and
  first successful use in 1985
• Allows massive amplification of specific
  sequences that have defined endpoints
• Fast, powerful, adaptable, and simple
• Many many many applications

usually
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Why amplify specific sequences?

• To obtain material for cloning and sequencing, or
  for in vitro studies
• To verify the identity of engineered DNA
  constructs
• To monitor gene expression
• To diagnose a genetic disease
• To reveal the presence of a micro-organism
• To identify an individual
• Etcetera, etcetera

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What you need for PCR

• Template DNA that contains the target sequence
• 2. Primers short oligonucleotides that define
  the ends of the target sequence
• 3. Thermostable DNA polymerase
• 4. Buffer, dNTPs
• 5. A thermal cycler

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A typical PCR program
Denaturation denature template strands (94C
for 2-5 minutes), can also add your DNA
polymerase at this temp. for a hot start
(adding DNA pol to a hot tube can prevent false
priming in the initial round of DNA
replication) Annealing The default is usually
55C. This temperature variable is the most
critical one for getting a successful PCR
reaction. This is the best variable to start with
when trying to optimize a PCR reaction for a
specific set of primers. Annealing temperatures
can be dropped as low as 40-45C, but
non-specific annealing can be a problem
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A typical PCR program
Extension generally 72C, this is the
operating temperature for many thermostable
DNA polymerases. Number of cycles Depends on
the number of copies of template DNA and the
desired amount of PCR product. Generally 20-30
cycles is sufficient.
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How it works a simple PCR reaction, first cycle
(Can also be Single-stranded)
94C
50C
Cycles of denaturation, primer annealing, and
primer extension by DNA polymerase
72C
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a simple PCR reaction, second cycle
like first cycle
new reactions
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a simple PCR reaction, third cycle
PCR animation http//www.dnai.org/b/index.html ht
tp//www.dnalc.org/ddnalc/resources/shockwave/pcra
nwhole.html
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Choosing primers

• Should be 18-25 (17-30?) nucleotides in length
  (giving specificity)
• Calculated melting temperature varies depending
  on the method used (55-65C using the Wallace
  Rule, eg. see MC), but should be nearly identical
  for both primers
• Avoid inverted repeat sequences and
  self-complementary sequences in the primers,
  avoid complementarity between primers (primer
  dimers)
• Have a G or C at the 3 end (a G/C clamp)
• Many computer programs exist for helping meet
  these criteria (ex Biology Workbench,
  workbench.sdsc.edu)

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Thermostable DNA polymerases
(See Molecular Cloning table 8-1)

• Isolated from thermophilic bacteria and archaea
  (T. aquaticus is a bacterium, not an archaeon)
• Bacterial enzymes (e.g. Taq) good for routine
  reactions and small PCR products, fidelity of
  replication is somewhat low
• Archaeal enzymes (e.g. Pfu) also good for routine
  reactions and best for cloning 3--5
  exonuclease activity provides very high fidelity,
  and enzymes are very stable to heat

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Thermal cyclers
Standard heat block, ramp times fairly long
(10 -20 seconds to change temperature), 30 cycle
PCR lasts 2-3 hours. Advantage easily
automated, heat blocks can PCR up to 384 samples
at a time Disadvantage relatively slow New
reactions are being sped up significantly --capil
lary tubes heated and cooled by blasts of air--30
cycle-PCR done in gt30 minutes (harder to scale
up) --fluid flow cells channels force liquid
through temperature gradients, very fast (but
still not widely available)
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Sources of problems in PCR

• Inhibitors of the reaction from the the template
  DNA preparation (protease, phenol, EDTA, etc)
• Cross-contamination by DNA from sources other
  than the template added
• if this becomes a problem
• Work in a laminar flow hood (decontaminate using
  UV light 254 nm)
• Use PCR dedicated pipettors (with barrier tips),
  PCR dedicated reagents
• Centrifuge tubes before opening them to prevent
  spattering, pipet contamination

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Controls to include in difficult PCRs
Bystander DNA not recognized by primers Target
DNA known to contain primer recognition sequences
Bystander DNA template DNA Target DNA Specific primers
Positive controls
1 -
2 - -
Negative controls
3 - - -
4 - -
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• Hot Start of PCR reactions
• Witholding some component of the reaction until
  the denaturing temperature is reached (94C)
• This helps prevent non-specific priming, which
  may occur at the low temperatures (room temp.) --
  the non-specific priming could give artifactual
  amplification as PCR block temperature rises
• Wait until 94C to add enzyme --or--
• Use enzyme bound to an inactivating enzyme
  antibody that releases at high temperature --or--
• Use wax beads containing Mg that can only be
  released at high temp.

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• Touchdown PCR
• Useful if your primers are not 100
  complementary to your template DNA (e.g.
  degenerate oligos), or when there are multiple
  members of the gene family you are amplifying
• Allows you to selectively amplify only the best
  sequences (with the least mismatches) while
  minimizing non-specific PCR products
• Start with 2 cycles at an annealing temperature
  about 3C higher than the calculated primer
  melting temperatures.
• Progressively reduce the annealing temperature
  by 1C at 2 cycle intervals

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Trouble-shooting
-- Very little product -- No PCR product --
Multiple bands Etc.
(see Molecular cloning, tables 8-4 and 8-5)
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III. Special applications for PCR A. Reverse
transcription PCR (for RNA measurements) B.
Quantitative (real-time) PCR C. PCR of long DNA
fragments D. Whole genome amplification E.
Inverse PCR E. MOPAC (mixed oligonucleotide
priming)
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Amplification of RNA (monitor gene expression)
reverse transcription PCR (RT-PCR)
Step 1 generate a 1st strand cDNA using reverse
transcriptase (catalyzes synthesis of DNA from an
RNA template)
A)
B)
C)
Step 2 normal PCR (from cDNA) using
gene-specific primers
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Quantitative(real time) PCR
The more target DNA there is, the more probe
anneals, the more it is cleaved (by Taqs 5-3
exonuclease activity)
Fluorescence measurements are done simultaneously
with PCR cycles, yields an instantaneous
measurement of product levels
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Quantitative Real Time (QRT) PCR
Position of the steep part of the curve varies
depending on the amount of template DNA or RNA,
can measure variation over 5 or 6 orders of
magnitude
more template
less template
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Another quantitative measure of double stranded
DNA in a PCR reaction binding of SYBR Green Dye
Non-fluorescing SYBR green dye
Fluorescing SYBR green dye
From the Molecular Probes website (www.probes.com)
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Use of a quenching dye to reduce measurement of
primer dimer artifacts in QRT-PCR
QSY quencher dye it absorbs fluorescence from
sybr green dyes in the vicinity--prevents
accumulation of signal from primer dimers
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Always do your controls! QRT-PCR using Sybr
green dye fluorescence
Standard curve what is threshold for specific
number of DNA molecules?
(From the Invitrogen website)
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PCR of long sequences (gt2 kb)

• Long DNAs are difficult to amplify
• Breakage of the DNA
• Non-processive behavior of DNA polymerase
• Misincorporation by error prone DNA polymerases

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PCR of long sequences (gt2 kb)

• Changes to protocol that assist in long PCR
• Make sure DNA is exceedingly clean
• Use DNA polymerase cocktail Taq for its high
  activity, and Pfu for its proofreading activity
  (it can actually correct Taqs mistakes)
• Increase time of extension reaction (5-20
  minutes, compared to the standard 1 minute for
  short PCRs)

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• Amplified product longer than 3 kilobases with
  high fidelity
• 10 times fewer mutations than with conventional
  PCR
• Taq DNA pol (no proofreading) plus an archaeal
  DNA pol (does proofreading)
• Betaine (amino acid analogue with several small
  tetraalkyammonium ions)--reduces non-specific
  amplification products--reduces non-complementary
  primer-template interactions? (unknown how it
  works)

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Whole genome amplification multiple displacement
amplification (MDA) Applications forensics,
embryonic disease diagnosis, microbial diversity
surveys, etc. How it works Strand-displacement
amplification used by rolling-circle replication
systems. Phi29 DNA polymerase (very low error
rate) and random hexamer primers, low
temperature! (30C)
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Whole genome amplification multiple
displacement amplification (MDA) 20-30
micrograms human DNA can be recovered from 1-10
copies of the human genome Distribution of
products appears to be random sampling of the
available template (and this is good!)
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Inverse PCR sequencing out from known sequence
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Vectorette PCR
First primer known sequence Vectorette primer
only in vectorette-ligated sequence--it cannot
anneal until there is a single round of primer
extension from the specific primer
http//www.bio.psu.edu/People/Faculty/Akashi/vectP
CR.html
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MOPAC Mixed oligonucleotide primed amplification
of cDNA

• If you only have a protein sequence, and you want
  to clone the gene for the protein
• Design oligonucleotides based on deduced mRNA
  (and DNA) sequence (but since multiple codons can
  encode the same amino acid, this gets complicated
  quickly)
• program your oligo synthesizer to make primer
  sets that are randomized for the degenerate
  positions of each codon
• use universal nucleotides like inosine, which
  base pairs with C, T, and A (limits degeneracy)
• Do your PCR and hope for the best