Experimental Techniques in a Nutshell

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Experimental Techniques in a Nutshell

• Rob Shields

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Overview of Talk

• Introduction
• Elementary Techniques
• Advanced Techniques

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Introduction

• Provide an overview of experimental techniques
  used in molecular biology
• Splicing of DNA
• Replication of DNA, RNA, Proteins
• Why do I need to know this?
• Understanding how experimental data was collected
  can play a part in how to analyze it

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Elementary Techniques

• Restriction Enzymes and Gel Electrophoresis
• Cloning Vectors and DNA Libraries
• 1D and 2D Protein Gels
• Hybridization and Blotting Techniques
• Protein Separation Techniques

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Restriction Enzymes

• Restriction Enzymes recognize short sequences (4
  to 8 bp long) of DNA and cut the molecule at this
  positions
• Blunt ends cut the sequence with no overhang
• Sticky ends cut the sequence with overhang

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Restriction Enzymes (Continued)

• Isoschizomers two restriction Enzymes that
  recognize the same sequence, but produce
  different cuts.

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Restriction Enzymes (Continued)

• Why are they so important anyway?
• Provided reproducible results
• Can analyze resulting fragments to detect
  mutations
• By choosing the correct set of enzymes one could
  create a set of fragments that is of interest to
  them (genes or region of DNA)

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Electrophoresis

• Applying a electric field to charge molecules in
  a gel
• The idea is to separate molecules based on their
  shape, size, and charge.
• The negatively charge DNA/RNA move from one side
  of the gel to the other.
• The size of the pores in the gel along with the
  size of the DNA/RNA determines how fast the
  DNA/RNA moves through the gel.
• In general, the smaller a molecule, the faster it
  moves.

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Electrophoresis (Continued)

• The length of the sequence being analyzed
  determines the gel type (smaller sequences need
  smaller pores).
• Pulse Field Electrophoresis is used for large
  sequences
• Pulses the electric field from side to side
• Makes the sequence zig-zag down the gel
• Dye is used so sequences appear under UV light

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Electrophoresis (Continued)
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Cloning Vectors

• Restriction fragment must be inserted to
  self-replicating element (plasmid or virus)
• Plasmids circular rings of DNA inside bacteria
• Replicating elements are called cloning vectors
• Plasmid and fragment must be cut with the same
  restriction enzyme both will have the same
  sticky end
• Sticky ends will (Hydrogen) bond together
  solidified by enzyme DNA ligase (covalent bonds)

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Cloning Vectors (Continued)

• Transformation insertion of cloning vectors to
  bacteria

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Cloning Vectors (Continued)

• A successful insert will inhibit a portion of the
  bacterias resistance to a given substance
• Placing copies of the colonies on a separate
  plate with the substance will kill the successful
  inserts
• Comparing the contents of both plates will
  determine the colonies of interest

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DNA Libraries

• Create cloning vectors for the entire genome of
  an organism used when a researcher does not
  know which fragment contains the gene of interest.

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DNA Libraries

• I size of insert GS genome size P
  probability fragment is represented by a clone in
  library (set at 99)

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DNA Library Problems

• Restriction Enzymes cut DNA regardless of
  start/end points of genes hence no guarantee a
  gene of interest fits on a single clone
• Indexing lots and lots of junk DNA that is
  useless

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cDNA Libraries

• cDNA complementary DNA formed from mRNA
• Contains only coding regions
• Tissue specific snapshot of gene expression
• Frequency represents expression level of
  corresponding gene
• Helps to determine intron/extron boundaries
• Gene expression at different parts of body
• Synthesize protein

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1 and 2D Protein Gels

• Charge of protein depends upon amount/type of
  amino acids
• Strong Detergent Sodium dodecyl sulfate (SDS)
• Sulfate group that binds to the hydrophobic
  portion of polypeptides
• Negative charge now proportional to size SDS
  bind points are proportional to number of amino
  acids
• Linear conformation
• Hydrophobic proteins can be inserted in gel
• Now simply a function of size

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1 and 2D Protein Gels

• Acrylamide monomers are polymerized to create
  polyacryamid gel
• The degree of cross linking is controlled -
  Allows for pore size control
• Mercaptoethanol is added to reduce sulfide
  bridges separates everything into single
  proteins

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1 and 2D Protein Gels

• Isoelectric Focusing use ph values to add
  second dimension solves problem of overlapping
  proteins

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Hybridization and Blotting Techniques

• Probe short fragment of DNA labeled so it can
  be visualized
• Target molecule the researcher wants to locate
• The probe is incubate with the target
• The probe locates the target and binds to it
  its label allows the researcher to easily locate
  the target molecule

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Southern Blotting

• After Electrophoresis the DNA must be separated
  from the gel onto a nitrocellulose or nylon
  surface
• Use capillary forces to move the DNA towards
  blotting paper
• Can locate modifications such as inserts and
  deletions if different probes are used can not
  locate point mutations

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Southern Blotting (Continued)
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Northern Blotting

• mRNA is used instead of DNA
• Different results in different areas of the body
• Can be used to see gene expression and protein
  levels

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Western Blotting

• Proteins are used directly instead of mRNA or DNA
• Antibody A is attach to the protein
• Antibody B is then inserted to bind to Antibody A
• Antibody B induces a chemical reaction that will
  be present under UV light

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In Situ Hybridization

• Allows for hybridization inside tissue
• Expose to high pH to separate the DNA double
  helix
• Probes bind directly to the DNA
• Allows researchers see how many copies of a gene
  exist
• Spatial and temporal studies can be conducted
• Location of elements within the cell allows for
  better understanding of why a protein does/does
  not function

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Centrifugation

• Fractionates molecules based on size and shape
• Sedimentation rate (Svedberg Units) S
• m mass density particle, sol ppar, psol
  friction f velocity v 82 angular
  velocity r distance from center

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Column Chromatography

• Column filled with solid carrier material and
  protein mixture placed on top
• The buffer proteins are held back at different
  rates by the solid carrier
• 4 Major Types
• By charge
• By Hydrophobicity
• Size
• Affinity chromatography
• Antibodies hold protein in place can be freed
  later

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Column Chromatography
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Advanced Techniques

• PCR (Polymerase Chain Reaction)
• DNA and Protein Chips
• Yeast Two-Hybrid System
• Mass Spectrometry
• Transgenic Animals
• RNA Inference

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PCR Polymerase Chain Reaction

• Primer is created such that it is complementary
  to the DNA both upstream and downstream
• DNA is heated to make it single stranded
• During the cooling phase the primer is added to
  the mixture and hybridize to the DNA
• Polymerase extends the primer, doubling the DNA
  fragment

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PCR (Continued)
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PCR (Continued)

• Also possible to copy mRNA
• Used for forensics
• Can be used to locate the corresponding gene to
  proteins

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DNA Chips

• DNA Microarrays
• Monitor the expression of multiple genes at the
  same time
• Constructed form a DNA library
• Fragments placed into individual cells on the
  chip
• mRNA extracted from samples we want to compare
• cDNA created
• cDNA incubated with chip shows clearly which
  genes are expressed

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DNA Chips
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Protein Chips

• Much more difficult to implement than DNA chips
• Size and shape of proteins vary greatly
• Interaction proteins/antibodies are various
• Each reaction might occur under different
  conditions

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Yeast Two-Hybrid System

• Used to detect protein-protein interactions
• Genes are fused to the DNA-binding or
  DNA-activating domain respectively of the TF
• Bait binds to the DNA, prey tries to express gene
• They interact if they are close enough together
  to simulate the expression of the gene

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Yeast Two-Hybrid System
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Mass Spectrometry

• Used to measure the mass of small molecules
  highly accurate
• Solvent is incubated with polymer in matrix
• Laser is point at the matrix solvent evaporates
• Matrix material is heated up which in turn
  charges the polymer the polymer is carried to
  the vapor phase
• The Time of Flight (TOF) from the matrix material
  to the end of a tube is recorded
• TOF sqrt(m/z) m mass z charge

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Transgenic Animals

• DNA Microinjection
• Chimera some cells develop with the injected
  DNA
• Random which cells with carry the injected DNA
  not favorable
• Embryonic stem cell-mediated transfer
• Inject stem cell with DNA
• Place stem cell within embryo of new animal
• Will grow with significant number of altered
  cells (down regulated)

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RNA Interference

• dsRNA double stranded RNA
• Recognized by DICER (endoribonuclease) cuts it
  into smaller sections (21-23 bp)
• Short fragments called siRNA overhang of 2 bp
  on 3 end
• SiRNA RNA-Induced Silencing Complex with proteins
  (RISC)
• If a complementary RNA is found it is cleaved
  in the middle and hence not able to express the
  gene

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RNA Interference
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Thank You

• Questions?

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References

• 1 Klipp, Herwig. Systems Biology in Practice
  Concepts, Implementation and Application. 2005.