Molecular Biology Techniques

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Molecular Biology Techniques
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Why Use Molecular Biology Techniques?

• Gene mutations can have serious consequences
• Dental diseases include amelogenesis and
  dentinogenesis imperfecta
• Also more severe diseases such as cystic fibrosis
  
• Molecular biology techniques can help to
  differentiate between normal genes and mutations

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Human Genome Project

• Elucidation of genes will allow for development
  of accurate diagnostics for treatment of diseases
  
• Single genes associated with cystic fibrosis and
  muscular dystrophy found
• Also discovery of diseases caused by several
  genes/gene interacting with environment

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Aims Objectives

• To examine the method and application of
• Restriction endonucleases
• Gene cloning and expression using plasmid cloning
  vectors
• Polymerase Chain Reaction
• Southern Blotting and nucleic acid hybridisation
• DNA sequencing

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Restriction Endonucleases

• Important tools in recombinant technology
• Have opened the way for gene cloning
• Endonucleases provide protection to bacterial
  cell against foreign DNA
• Bacterias own DNA is modified by methylation of
  bases

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• Enzymes are named from bacterial species that
  they are derived from
• EcoRI ? is the first enzyme to be isolated from
  E. coli strain RY13
• The enzymes recognise 4-6bp of DNA and cleave
  phosphodiester bond

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• Restriction site is palindrome ?
• EcoRI
• forward 5-3 GAATTC
• reverse 3-5 CTTAAG
• Digestion with EcoRI gives staggered end other
  enzymes produce blunt ends ?
• EcoRI HaeIII
• G AATTC GG CC
• CTTAA G CC GG

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• Restricted fragments can be separated by size
  using gel electrophoresis
• Standard markers run alongside used to size them
• Polyacrylamide gels used to separate low
  molecular weight molecules
• Agarose gels used to separate out high molecular
  weight molecules

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Plasmids and Gene Cloning

• Extrachromosomal DNA molecules
• Confer antibiotic resistance
• Vectors are plasmids designed for gene cloning
• 3 basic requirements of vectors
• ability to self replicate within host
• possession of selection marker
• must be capable of being digested

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• When vector and DNA are digested with the same
  enzymes both can be joined together using DNA
  ligase
• DNA ligase catalyses formation of phosphodiester
  bond
• Resulting recombinant DNA can be introduced into
  cells treated to make them permeable

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• As cells divide and grow, recombinant plasmid
  replicates many times
• Relevant clones can be selected by use of
  antibiotic within media
• Only DNA lt10kb can be inserted into vectors

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Polymerase Chain Reaction

• Amplifies DNA molecules exponentially using DNA
  polymerase
• Only flanking sequence of DNA needs to be known
  for design of primers
• Primers are short oligonucleotides (20-35)
  representing forward and reverse sequences
• Each primer anneals to 3 portion of the DNA
  template

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Recommended Reading

• Biochemistry (2002) Stryer
  6
• Biochemistry (1995) Voet and Voet
  28
• Mol. Cell Biology (2000) Lodish et al.
  7
• Mol. Biology of the Cell (2002) Alberts et al.
  7
• Harpers Biochemistry (23rd ed.) Murray et al.
  42
• Instant Notes in Biochem. (1997) Hames et
  al. I
• Instant Notes in Mol. Biol. (1997) Turner et al.
  G/H/I/J
• Biochemistry (Mols., Cells and the Body) Dow
  et al. -
• Lippincotts Illus. Reviews (2nd ed.) Champe and
  Harvey 33

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Aims Objectives

• To examine the method and application of
• Restriction endonucleases
• Gene cloning and expression using plasmid
  cloning vectors
• Polymerase Chain Reaction
• Southern Blotting and nucleic acid hybridisation
• DNA sequencing

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Southern Blotting

• Used to detect copy number of gene
• Can also detect gross alterations in a gene
• Allows for visualisation of specific DNA fragment
  among thousands of molecules
• Can probe for more than one fragment of DNA

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Southern Blot Procedure

• Digested DNA with appropriate restriction enzyme
• Run fragments on electrophoresis gel to separate
• Denature DNA to make single strands and transfer
  to nitrocellulose membrane
• Hybridise DNA with specific probe
• Detect DNA fragment

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Hybridisation of Nucleic Acids

• Heating causes DNA to denature into single
  strands they can re-anneal when temperature
  lowered
• For annealing to occur require sufficient
  complementary nucleotide sequence between the two
  nucleic acid molecules
• Annealing can be achieved with any two single
  stranded nucleic acid molecules

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• Hybridisation can be used to establish success of
  cloning experiment
• For RNA it can be used as an indication of the
  level of gene expression
• Specific probe can be DNA fragment, cloned DNA or
  synthetic oligonucleotide
• Labelling of probe is usually with 32P
• Same basic method for all forms of nucleic acids

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DNA Sequencing

• Sanger technique used frequently these days
• Uses dideoxynucleotides to terminate chain
  elongation during DNA synthesis
• Radioactive nucleotide incorporated into
  elongating strands
• Four reactions carried out simultaneously,
  containing 4dNTPs and single ddNTP

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Extension of Polynucleotide Chain
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• Extension reaction products are resolved by gel
  electrophoresis
• Results obtained when gel is dried and exposed to
  X-ray film
• Bands near bottom of gel represent short reaction
  products those at top the longest
• The process is now automated and uses fluorescent
  dye

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Sanger Technique for Sequencing
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Recommended Reading

• Biochemistry (2002) Stryer
  6
• Biochemistry (1995) Voet and Voet
  28
• Mol. Cell Biology (2000) Lodish et al.
  7
• Mol. Biology of the Cell (2002) Alberts et al.
  7
• Harpers Biochemistry (23RD Ed.) Murray et al.
  42
• Instant Notes in Biochem. (1997) Hames et
  al. I
• Instant Notes in Mol. Biol. (1997) Turner et al.
  G/H/I/J
• Biochemistry (Mols., Cells and the Body) Dow
  et al. -
• Lippincotts Illus. Reviews (2nd Ed.) Champe and
  Harvey 33