Practical molecular biology

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Practical molecular biology

• Dr. Alexei Gratchev
• Prof. Dr. Julia Kzhyshkowska
• Prof. Dr. W. Kaminski

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Course structure

• 08.10 Plasmids, restriction enzymes, analytics
• 09.10 Genomic DNA, RNA
• 10.10 PCR, real-time (quantitative) PCR
• 11.10 Protein analysis IHC
• 12.10 Flow cytometry (FACS)

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Nucleic acid chemistry

• DNA (deoxyribonucleic acid) and RNA (ribonucleic
  acid) store and transfer genetic information in
  living organisms.
• DNA
• major constituent of the nucleus
• stable representation of an organisms complete
  genetic makeup
• RNA
• found in the nucleus and the cytoplasm
• key to information flow within a cell

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Why purifying genomic DNA?

• Many applications require purified DNA.
• Purity and amount of DNA required (and process
  used) depends on intended application.
• Example applications
• Tissue typing for organ transplant
• Detection of pathogens
• Human identity testing
• Genetic research

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DNA purification challenges

• Separating DNA from other cellular components
  such as proteins, lipids, RNA, etc.
• Avoiding fragmentation of the long DNA molecules
  by mechanical shearing or the action of
  endogenous nucleases.Effectively inactivating
  endogenous nucleases (DNase enzymes) and
  preventing them from digesting the genomic DNA is
  a key early step in the purification process.
  DNases can usually be inactivated by use of heat
  or chelating agents.

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DNA purification

• There are many DNA purification methods. All
  must
• Effectively disrupt cells or tissues(usually
  using detergent)
• Denature proteins and nucleoprotein complexes(a
  protease/denaturant)
• Inactivate endogenous nucleases(chelating
  agents)
• Purify nucleic acid target away from other
  nucleic acids and protein(could involve RNases,
  proteases, selective matrix and alcohol
  precipitations)

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Denaturing agents

• Ionic detergents, such as SDS, disrupt
  hydrophobic interactions and hydrogen bonds.
• Chaotropic agents such as urea and guanidine
  disrupt hydrogen bonds.
• Reducing agents break disulfide bonds.
• Salts associate with charged groups and at low or
  moderate concentrations increase protein
  solubility.
• Heat disrupts hydrogen bonds and nonpolar
  interactions.
• Some DNA purification methods incorporate
  proteases such as proteinase K to digest
  proteins.

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DNA isolation

• DNA must be separated from proteins and cellular
  debris.
• Separation Methods
• Organic extraction
• Salting out
• Selective DNA binding to a solid support

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Organic extraction

• DNA is polar and therefore insoluble in organic
  solvents.
• Traditionally, phenolchloroform is used to
  extract DNA.
• When phenol is mixed with the cell lysate, two
  phases form. DNA partitions to the (upper)
  aqueous phase, denatured proteins partition to
  the (lower) organic phase.
• DNA is a polar molecule because of the negatively
  charged phosphate backbone.
• This polarity makes it more soluble in the polar
  aqueous phase.

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Genomic DNA isolation phenol 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
• Genomic DNA is isolated as pieces up to 1 Mbp!

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Genomic DNA isolation phenol extraction
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Binding to a support material

• Most modern DNA purification methods are based
  on purification of DNA from crude cell lysates by
  selective binding to a support material.
• Support Materials
• Silica
• Anion-exchange resin
• Advantages
• Speed and convenience
• No organic solvents
• Amenable to automation/miniaturization
• Disadvantage
• DNA fragmentation

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Silica matrix based genomic DNA isolation
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Genomic DNA analysis
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Concentration measurement
Photometric measurement of DNA concentration UV
260 nm Conc50xOD260
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Total cellular RNA

• Messenger RNA (mRNA) 1-5
• Serves as a template for protein synthesis
• Ribosomal RNA (rRNA) gt80
• Structural component of ribosomes
• Transfer RNA (tRNA) 10-15
• Translates mRNA information into the appropriate
  amino acid

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What RNA is needed for?
Messenger RNA synthesis is a dynamic expression
of the genome of an organism. As such, mRNA is
central to information flow within a cell.
Size examine differential splicing Sequence
predict protein product Abundance measure
expression levels Dynamics of expression
temporal, developmental, tissue specificity
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RNA isolation
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RNA purification

• Total RNA from biological samples
• Organic extraction
• Affinity purification
• mRNA from total RNA
• Oligo(dT) resins
• mRNA from biological samples
• Oligo(dT) resins

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Total RNA purification

• Goal Isolate RNA from cellular components
• Cells or tissue must be rapidly and efficiently
  disrupted
• Inactivate RNases
• Denature nucleic acid-protein complexes
• RNA selectively partitioned from DNA and
  protein
• Isolation from different tissues/sources raises
  different issues

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RNases

• RNases are naturally occurring enzymes that
  degrade RNA
• Common laboratory contaminant (from bacterial
  and human sources)
• Also released from cellular compartments during
  isolation of RNA from biological samples
• Can be difficult to inactivate

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Protecting agains RNases

• Wear gloves at all times
• Use RNase-free tubes and pipet tips
• Use dedicated, RNase-free, chemicals
• Pre-treat materials with extended heat (180C
  for several hours), wash with DEPC-treated water,
  NaOH
• or H2O2
• Supplement reactions with RNase inhibitors
• Include a chaotropic agent (guanidine) in the
  procedure
• Chaotropic agents such as guanidine inactivate
  and precipitate RNases and other proteins

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Organic extraction of total RNA
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Organic extraction of total RNA

• Advantages
• Versatile - compatible with a variety of sample
  types
• Scalable - can process small and large samples
• Established and proven technology
• Inexpensive
• Disadvantages
• Organic solvents
• Not high-throughput
• RNA may contain contaminating genomic DNA

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Affinity purification of total RNA
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Affinity purification of total RNA

• Advantages
• Eliminates need for organic solvents
• Compatible with a variety of sample types
  (tissue, tissue culture cells, white blood cells,
  plant cells, bacteria, yeast, etc.)
• DNase treatment eliminates contaminating genomic
  DNA
• Excellent RNA purity and integrity

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RNA analysis
Photometric measurement of RNA concentration UV
260 nm Conc40xOD260
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Alternative nucleic acids purification protocol

• CsCl gradient centrifugation
• The best quality of RNA
• RNA and DNA isolated simultaneously
• has a 36h centrifugation step

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Questions?