DNA and RNA isolation and purification (course readings 10 by zwk61917

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									     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
cell growth                                and lysis




              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                           N+       Br-
 conditions)
                                CH3
                                           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
cell growth                                and lysis




              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 guanidinium-
   treated 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.          Theory of PCR
II.         Components of the PCR reaction
III.        A few advanced applications of PCR

       a)     Reverse transcription PCR (for RNA
              measurements)
       b)     Quantitative real-time PCR
       c)     PCR of long DNA fragments
       d)     Inverse PCR
       e)     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
                         Cycles of
                         denaturation,
                         primer annealing,
                72°C     and primer
                         extension by DNA
                         polymerase
        a simple PCR reaction, second cycle




     new                                 like
reactions                                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   template   Target   Specific   Bystander DNA:
           DNA         DNA        DNA      primers    not recognized
Positive                                              by primers
controls
                                                      Target DNA:
1          +           -          +        +          known to
                                                      contain primer
2          -           -          +        +          recognition
Negative                                              sequences
controls

3          -           -          -        +
4          +           -          -        +
            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
                    more
measure             template   less
variation over 5               template

or 6 orders of
magnitude
 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 rolling-
circle 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

								
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