Blotting Techniques

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Blotting Techniques
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Northern and Southern blotting

• Understanding the terminology
• 6 steps of a blot experiment
• Sample preparation and gel
• Transfer
• Blot fixation
• Making a probe
• Probe hybridization
• Blot visualization
• Experimental interpretation the pros and cons
  of blots

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Southern blots

• Named after British biochemist Edwin Southern
  (alive and publishing!)
• Southern EM. Detection of specific sequences
  among DNA fragments separated by gel
  electrophoresis.J. Mol Biol. 1975 Nov
  598(3)503-17.
• A technique for identifying specific sequences
  of DNA in which DNA fragments are separated by
  electrophoresis, transferred to a membrane, and
  identified with a suitable probe.
• Detects restriction fragments following a
  restriction enzyme digest of genomic DNA (RFLPs)
• Detects RT-PCR fragments
• Can be used to screen cDNA libraries

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Northern blots

• Name is derived from a LAME scientific sense of
  humor a word play on the term Southern blot.
  The term Western blot also has this origin.
• A technique for identifying specific sequences of
  RNA (usually mRNA) in which RNA molecules are
  separated by electrophoresis, transferred to a
  membrane, and identified with a suitable probe
  (RNA or cDNA)
• Detects mRNA molecules

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Summary of procedure

• Run samples on a gel
• Transfer to a membrane
• Link sample to membrane
• Make a probe
• Hybridize probe to membrane
• Detect probe

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Sample preparation - Southern

• genomic DNA is HUGE too big to enter an agarose
  gel
• Use a restriction enzyme to break it into smaller
  pieces (also generates sequence specific patterns
  mutations alter these patterns)
• Buffer used is 1X TAE or 0.5X TBE

smear of digested DNA
Agarose gel
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Sample preparation - Northern

• RNA can be difficult to work with. Always wear
  gloves and use RNAase free solutions
  (DEPC-treated water) and glassware (oven baked
  for at least 2 hours at 180)
• AUTOCLAVING DOES NOT REMOVE RNAase!!!!!!
• Use polyA enriched mRNA for maximum sensitivity
• Formamide and formaldehyde in the loading buffer
  and gel prevent formation of RNA secondary
  structure
• Gel buffer is 1x MOPS
• For both Northerns and Southerns, at least 1 lane
  should contain molecular weight markers

Formaldehyde gel
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Setting up a transfer

• Preparing the gel for transfer
• For Southerns, DNA should be depurinated (nicked)
  w/ HCl to facilitate sticking to membrane , base
  denatured w/ NaOH to make it single stranded, and
  neutralized afterwards
• For Northerns, gel should be soaked in transfer
  buffer to remove formaldehyde (it inhibits
  transfer)
• Transfer buffer 20X SSC (NaCl and Na citrate).
  Especially important if you are NOT using a
  charged membrane.

Important points Preparing the gel for
transfer Proper transfer setup
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Setting up a transfer assembly

• Order of assembly from bottom to top
• Wick? inverted gel?membrane?filter paper?paper
  towels?weight
• Transfer proceeds via capillary action, ie simple
  diffusion
• Correcting for the mirror image Keeping track
  of where your samples are!!!

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Keys to a successful transfer

• No air bubbles
• Roll out w/ a pipet
• No short circuits
• Use parafilm or old xray film to create a mask
• Plenty of paper towels
• Time
• Good to set up just before you leave and let it
  transfer overnight (or at least 6 hours)

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Post transfer issues

• After a transfer, DNA or RNA is stuck to the
  membrane, right? WRONG!
• Following transfer, you must immediately fix the
  sample to the membrane by
• Baking (120 for a 30 minutes)
• UV crosslinking (using a crosslinker)
• After crosslinking, the blot can be stored for
  years (dry at -80)

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UV crosslinker
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Making a probe

• A probe is a copy of a DNA sequence that
  contains a detectable label

YFG
Labeling reaction
Primers
YFG
YFG
YFG
Labeled probes
YFG
YFG
Template
YFG

• The template is usually made by PCR
• For Southerns, the template should be made from
  genomic DNA, ie introns and exons Why?
• For Northerns, the template should be made from
  cDNA, ie exon sequence only Why?

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2 kinds of label

• Non-radioactive
• Digoxigenin
• Biotin
• Radioactive
• 32P

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Types of labeling methods

• Non-radioactive
• Digoxigenin (DIG)
• The DIG is chemically linked to dUTP It is
  incorporated into the random hexamer-primed
  fragments
• Use an antibody to detect the DIG after
  hybridization (just like a Western blot

Advantages Safety Short detection times -
usually seconds to minutes Stability Once
made, a probe can be used for a couple of years
later Cost In the long term, very inexpensive
compared to other methods Disadvantages Less
sensitive Detection requires extra steps Must
physically remove probe through a harsh stripping
procedure in order to use the blot again
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Types of labeling methods

• Radioactive
• 32P
• A phosphate in most dCTPs is radiolabeled
• The radioactive beta particles emitted are
  detectable with xray film
• Advantages
• Sensitivity
• Ease of reprobing just let bound probe decay
• Disadvantages
• Lots of safety precautions required
• Unstable
• Long detection times (sometimes weeks!)
• Expensive

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Making a probe

• Commonly used labeling methods
• Nick translation
• PCR labeling
• Random hexamer labeling easiest and most
  commonly used
• Purity of probe depends on purity of template
• Clean templatepure probeclean results!

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Labeling reaction

• Method for generating 32P labeled and Dig
  labeled probes is identical
• Purify labeled probes away from unincorporated
  label by a precipitation step or column
  filtration
• Quantifying the amount of labeled probe is
  critical
• add too much and your blot looks like
• add to little and your blot looks like
• 32P probe - 1-5 ng/ml
• Dig probe 20-50 ng/ml

Dig
YFG
dATP, dUTP, dGTP, dTTP
Random hexamers
YFG
YFG
YFG
Pure gDNA or cDNA template
DNA polymerase I Enzyme buffer 37 for at least 1
hour
YFG
Dig labeled probes
YFG
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Hybridization

• Prehybridization
• The blot is mostly empty space your probe will
  easily bind to this empty space NOT GOOD!
• Prehybridization fills this empty space with
  non-specific nucleic acid usually salmon sperm
  DNA (if youre studying salmon or a related fish,
  you will need to find another blocking agent!)
• Usually 1 hour at the hybridization temperature
  is sufficient
• Hybridization usually goes overnight
• Usually done in a hybridization oven
• Whats in the hybridiziation solution?
• Formamide displaces water opens nucleic acid
• Denhardts reagent blocking solution (BSA, PVP,
  Ficoll)
• Dextran sulfate artificially increases probe
  concentration
• SDS alleviates hydrophobic interaction 2
  nucleic acids can stick together simply through
  hydrophobic interactions
• Hybridization solutions can be purchased much
  easier and more cost effective than making all
  this stuff yourself!

Once the hybridization has been started, DO NOT
let it dry out! Bound probe can not be removed
if the blot has dried!
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Hybridization oven
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Hybridization

• Stringency
• High stringency Probe wont bind anything
• Low stringency Probe binds everything
• Things that affect stringency
• Salt (NaCl) concentration (lower high
  stringency)
• Formamide concentration (higher high
  stringency)
• Temperature of incubation (higher high
  stringency)
• Finding the correct stringency conditions can
  vary from probe to probe
• Using a commercially available hyb solution, a
  good place to start is
• 68 for an RNARNA hybridizations
• 50 for DNARNA hybridizations
• 42 for DNADNA hybridizations
• Post hybridization washes
• Gets rid of excess unbound probe
• Contains low salt and high detergent

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Detection

• If you used a 32P probe, you can now expose the
  blot to film with an intensifying screen
  amplifies signal at low temperature ? put your
  blot in the -80 freezer.
• Dig probes require a few more steps before you
  can see them

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Detecting the Dig label
Light

• Anti-Dig antibody coupled to the alkaline
  phosphatase (AP) enzyme ? PROTEIN
• Nylon loves to bind protein
• Block with a BSA solution (30 minutes)
• Incubate w/ antibody (30 minutes)
• Wash (30 minutes)
• AP dephosphorylates AMPPD- Its destabilization
  gives off 477nm light
• Film detects light
• Detection is done in a film cassette in a dark
  room

AP
Blot
Bound Dig-labeled probe
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Applications of a Southern blot

• Genotyping
• Is your knockout really a knockout?
• Genetic deficiency mapping
• SNP mapping (DNA fingerprinting)
• Variations include FISH

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Applications of a Northern blot

• mRNA detection
• Where is the gene expressed?
• Which conditions alter mRNA levels?
• How big is the gene?
• Is there more than one isoform?
• Variations ISH, IHC

microarray
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Northern interpretation

• Advantages
• Reveals the size of the transcript (including all
  5 and 3 UTR)
• Detects alternatively spliced isoforms
• Indicates the relative abundance of these
  isoforms
• Disadvantages
• Northerns have relatively low sensitivity
• Cant clone for further analysis
• No information about rate of transcription

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Visualizing blots Summary

• Run samples on a gel
• Transfer to a membrane
• Link sample to membrane
• Make a probe
• Hybridize probe to membrane
• Detect probe

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Further information

• monsheel.sodhi_at_vanderbilt.edu
• Roche website
• http//www.roche-applied-science.com/fst/products.
  htm?/prod_inf/manuals/dig_man/dig_toc.htm