Download What do we need DNA for?

DNA and RNA isolation and purification (course
readings 10 and 11)
I.
Genomic DNA preparation overview
II.
Plasmid DNA preparation
III.
DNA purification
• Phenol extraction
• Ethanol precipitation
IV.
RNA work
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?
DNA purification: overview
cell harvest
and lysis
cell growth
DNA concentration
DNA purification
Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents
• Organic solvent
• Proteases
(lysozyme)
• Heat
“cell extract”
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
2 ways to concentrate the genomic DNA
70% final conc.
“spooling”
Ethanol precipitation
Genomic DNA prep in plants -how get rid of carbohydrates?
CTAB:
Cationic
detergent
CH3
CH3
(low ionic
conditions)
N+
CH3
Br-
C16H33
(MC 6.61-6.62)
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
Plasmid purification: alkaline lysis
Alkaline
conditions
denature
DNA
Neutralize:
genomic DNA
can’t renature
(plasmids
CAN because
they never
fully
separate)
DNA purification: silica binding
Binding occurs in presence of high
salt concentration, and is disrupted
by elution with water
DNA purification: phenol/chloroform extraction
1:1 phenol : chloroform
or
25:24:1 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
Phenol extraction
1. Aqueous volume (at least 200 microliters)
2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube
4. Repeat steps 2 and 3 until there is no precipitate at
phase interface
5. (extract aqueous layer with 2 volumes of chloroform)
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)
DNA purification: overview
cell harvest
and lysis
cell growth
DNA concentration
DNA purification
DNA -------------->
mRNA
--------------> 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
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
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
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, 300°C, 4 hours
USE “RNase-free” PIPET TIPS
TREAT EQUIPMENT WITH DEPC
Top 10 sources of RNAse contamination
(Ambion Scientific website)
1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)
10) Enzyme preparations
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)
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
Making and using mRNA (1)
Making and using mRNA (2)
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 (-80°C)
in formamide (deionized) at -20°C
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
Other methods to capture mRNA
Poly(U) sepharose chromatography
Poly(U)-coated paper filters
Streptavidin beads:
•A biotinylated oligo dT is added to guanidiniumtreated cells, and it anneals to the polyA tail of mRNAs
•Biotin/streptavidin interactions permit isolation of the
mRNA/oligo dT complexes
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
In vitro amplification of DNA by PCR
I.
II.
III.
Theory of PCR
Components of the PCR reaction
A few advanced applications of PCR
a)
b)
c)
d)
e)
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
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
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
What you need for PCR:
1. 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
A typical PCR program:
Denaturation: denature template strands (94°C 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 55°C. 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 40-45°C,
but non-specific annealing can be a problem
A typical PCR program:
Extension: generally 72°C, 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.
How it works:
a simple PCR reaction, first cycle
(Can also be
Single-stranded)
94°C
50°C
72°C
Cycles of
denaturation,
primer annealing,
and primer
extension by DNA
polymerase
a simple PCR reaction, second cycle
new
reactions
like
first
cycle
a simple PCR reaction, third cycle
PCR animation:
http://www.dnai.org/b/index.html
http://www.dnalc.org/ddnalc/resources/shockwave/pcran
whole.html
Choosing primers:
• Should be 18-25 (17-30?) nucleotides in length (giving
specificity)
• Calculated melting temperature varies depending on the
method used (55-65°C 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)
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
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
--capillary tubes heated and cooled by blasts of air-30 cycle-PCR done in >30 minutes (harder to scale up)
--fluid flow cells: channels force liquid through
temperature gradients, very fast (but still not widely
available)
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
Controls to include in difficult PCRs:
Bystander
DNA
template
DNA
Target
DNA
Specific
primers
Positive
controls
1
+
-
+
+
2
-
-
+
+
3
-
-
-
+
4
+
-
-
+
Negative
controls
Bystander DNA:
not recognized
by primers
Target DNA:
known to
contain primer
recognition
sequences
Hot Start of PCR reactions
•
•
Witholding some component of the reaction until the
denaturing temperature is reached (94°C)
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
A) Wait until 94°C to add enzyme --or-B) Use enzyme bound to an inactivating enzyme
antibody that releases at high temperature --or-C) Use wax beads containing Mg++ that can only be
released at high temp.
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 3°C
higher than the calculated primer melting temperatures.
• Progressively reduce the annealing temperature by 1°C at 2
cycle intervals
Trouble-shooting:
-- Very little product
-- No PCR product
-- Multiple bands
Etc.
(see Molecular cloning,
tables 8-4 and 8-5)
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)
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
Quantitative
(real time)
PCR
The more target DNA there
is, the more probe anneals,
the more it is cleaved (by
Taq’s 5’-3’ exonuclease
activity)
Fluorescence measurements
are done simultaneously with
PCR cycles, yields an
instantaneous measurement of
product levels
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
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)
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
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)
PCR of long sequences (>2 kb)
Long DNAs are difficult to amplify
– Breakage of the DNA
– Non-processive behavior of DNA
polymerase
– Misincorporation by error prone DNA
polymerases
PCR of long sequences (>2 kb)
Changes to protocol that assist in long PCR
– Make sure DNA is exceedingly clean
– Use DNA polymerase “cocktail”: Taq for it’s
high activity, and Pfu for its proofreading
activity (it can actually correct Taq’s
mistakes)
– Increase time of extension reaction (5-20
minutes, compared to the standard 1
minute for short PCRs)
•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)
Whole genome amplification: multiple
displacement amplification (MDA)
Applications: forensics, embryonic disease diagnosis,
microbial diversity surveys, etc.
How it works:
Strand-displacement amplification used by rollingcircle replication systems.
Phi29 DNA polymerase (very low error rate) and
random hexamer primers, low temperature! (30°C)
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!)
Inverse PCR:
sequencing
“out” from
known
sequence
“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/vectPCR.html
MOPAC: Mixed oligonucleotide primed
amplification of cDNA
If you only have a protein sequence, and you want to clone
the gene for the protein:
1. Design oligonucleotides based on deduced mRNA (and
DNA) sequence (but since multiple codons can encode
the same amino acid, this gets complicated quickly)
2. program your oligo synthesizer to make primer sets that
are randomized for the degenerate positions of each
codon
3. use universal nucleotides like inosine, which base pairs
with C, T, and A (limits degeneracy)
4. Do your PCR and hope for the best