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M sc2
 
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M sc2 M sc2 Presentation Transcript

  • Molecular Approaches to Nutrition
    • Molecular Biology 2
    • Principles and Methods Dr. Janice Drew
  • Principles and Methods
    • Purification and handling of DNA/RNA
    • Gel Electrophoresis
    • Nucleic Acid Hybridisation
    • Cutting and rejoining DNA
    • Methods of introducing DNA into cells
    • PCR
    • DNA sequencing
    • Sequence interpretation
  • Handling of DNA/RNA
    • DNases and RNases
    • Glass and plasticware
    • Solutions
  • Extraction of DNA/RNA
    • DNA extraction
    • Alkaline lysis
    • Neutralisation
    • Precipitation of proteins and cell debris
    • Precipitation or elution using spin column
    • RNA extraction
    • Lysis incorporating instantaneous inactivation of RNases
    • Separation of contaminating DNA
    • Precipitation or elution using spin column
  • Quantitation and analysis of DNA/RNA
    • Spectrometric determination at 260nm
    • Gel Electrophoresis
    • Agilent technology
  • Gel Electrophoresis
    • Nucleic acids are negatively charged
      • PO 4 - groups
    • Electrophoresis resolves by size
    • Agarose is the usual gel matrix
    • Ethidium bromide/SYBR green ‘stains’ DNA & RNA
      • Fluorescent colour under UV illumination
  • Agarose Gel Preparation Agarose : fine white powder; polysaccharide (galactose polymer) isolated from seaweed. 1% (w/v) dissolves in Tris-acetate buffer at ~60 ° C and the solution sets at ~30 ° C
  • Agarose Gel Image
  • Agilent Technology
  • Electropherogram showing Agilent analysis of total RNA 18S 28S Times (seconds) Fluorescence
  • Hybridisation - Identification of DNA/RNA
    • Agarose gel electrophoresis separates nucleic acids on the basis of size - does not identify DNA/RNA fragments
    • Nucleic acid probes are used to identify specific DNA/RNA sequences in a gel
    • Probe is a known nucleic acid sequence
    • Relies on the principle of base pairing - complementary DNA/RNA sequences stick (hybridise) together
  • Hybridisation - Identification of DNA/RNA
    • Many molecular biology procedures to identify specific DNA/RNA sequences use this principle -
    • Southern (DNA) or Northern (RNA) blotting
    • In situ hybridisation
    • Microarrays
    • Antisense technologies
  • Probe Production
    • Synthesise a known fragment
    • OR
    • Purify a known fragment of DNA
      • Restriction enzyme digestion
    • Heat denature to give single strands
    • Add primers, DNA polymerase and radioactive/colour labelled nucleotides
      • Make a radioactive/ colour labelled complementary strand
    • Denature to give single strands
  • HYBRIDISATION OVEN Incubate filter and probe in hybridisation buffer TREAT and BLOT GEL Transfer to nylon membrane nylon membrane and transferred DNA Southern/Northern Blotting and Hybridisation
  • Restriction Endonucleases
    • Restriction endonucleases cut DNA
    • Present in bacteria
    • Cut at sequence specific sites
      • Usually 4 or 6 base pairs long
    • Bacteria use them to destroy ‘foreign’ DNA
      • Bacteria protect their own DNA against self-cutting by special methylation of their DNA
    • Restriction enzymes can be purified and are used in genetic engineering studies
  • Restriction Endonucleases
    • Example Restriction enzymes
      • Eco R I ( E . co li R estriction Endonuclease I )
      • Stu I ( S treptomyces tu bercidicus I)
    GAATTC CTTAAG 5’ 5’ 3’ 3’ Sticky Ended Blunt Ended AGGCCT TCCGGA 5’ 5’ 3’ 3’ Eco R I Stu I Palindromic Axis of rotational symmetry
  • Molecular Scissors and Glue
    • There are 100’s of restriction enzymes, each one with a different recognition site
      • These enzymes are ‘molecular scissors’ and can be used to specifically cut long DNA strands into smaller pieces
    • The T4 virus, which infects E. coli , has an enzyme, T4 DNA ligase, which can form a phosphodiester bonds between DNA molecules
      • Purified T4 DNA ligase can be used as ‘molecular glue’ to join pieces of DNA. This enzyme is widely used for DNA cloning
  • Ligation of DNA Eco R I OH 3’ 5’ PO 4 T4 DNA ligase catalyses the formation of phosphodiester bonds PO 4 5’ 3’ OH T4 DNA Ligase T4 DNA Ligase Stu I Circular DNA Eco R I G CTTAA AATTC G
  • Methods of introducing DNA into cells
    • Plasmids
    • Viruses
    • DNA and RNA viruses
    • Phage vectors
  • Cloning DNA into Plasmids
    • Bacteria have a circular DNA genome
      • 5 to 10 million base pairs (bp) in size
    • Many bacteria also contain plasmids
      • Small circular DNA molecules, ~3,000 to 50,000 bp
      • Note : The bacterial genome is not a plasmid
    • Plasmids contain ‘extra’ genes which are often vital for the survival of the bacterium
      • Nutrient metabolism, antibiotic resistance
    • Plasmids can be used as vectors in which foreign DNA can be ligated (cloned)
  • A General Laboratory Plasmid Multiple Cloning Site A foreign gene can be ligated into a plasmid, and the genetically engineered plasmid introduced into E. coli .
  • Cloning DNA into a Plasmid Both plasmid and foreign DNA have sticky Eco R I ends Insertion into E. coli (transformation) Agar plates contain antibiotic. Grow at 37 °C Place 1 colony in liquid media + antibiotic. Grow at 37 °C Purify Plasmid DNA (Billions of copies)
  • DNA and Retroviruses can serve as vehicles for the introduction of new DNA into a cell
  • DNA / RNA viruses as ‘vehicles’ Chromosomal DNA Viral DNA Integration into genome gene x Gene Therapy and Transgenics
  • Polymerase Chain Reaction (PCR)
    • PCR generates multiple copies of DNA
      • Heat resistant DNA polymerase used to copy a section of DNA e.g Taq
    • Very efficient copying
      • Billions of copies from a single ‘template’ DNA
    • Small volume / quick analysis
  • Polymerase Chain Reaction (PCR)
    • Entire reaction performed in single tube
      • 10 to 50 μ l volume
    • Reaction contains
      • Template DNA, heat resistant DNA polymerase, a pair of specific DNA primers (in excess over the template), nucleotide bases, appropriate reaction buffer
    • Reaction is repeatedly cycled through 3 temperatures (x30)
      • 95 ° C (makes DNA single stranded)
      • ~55 - 60 ° C (primers anneal to template DNA)
      • 72 ° C DNA polymerase copies DNA, starting from the primers
  • A Thermocycler This thermocycler can accept 1500 reactions at a time, and complete them in 2 to 4 hours.
  • Principal of PCR A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 5 ’ 3 ’ 5 ’ 3 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 5 ’ 3 ’ | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T o o l t o 5 5 ° C Cool. This allows specific ‘ primers’ to anneal as shown DNA (Double Stranded) Heat Denature (Becomes Single Stranded) Heat to 72 °C. Heat resistant DNA polymerase extends new DNA from the primers Heat to 72 °C C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | G T A T G 5 ’ 3 ’ G T T G C | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ H e a t 9 5 ° C ( D e n a t u r e s ) A d d S p e c i f i c P r i m e r s C
  • DNA Sequencing
    • A specific primer binds to denatured DNA
    • Heat resistant DNA polymerase extends a new strand from this primer
    • Complementary nucleotides are added as appropriate
    • In the reaction are small quantities of coloured dideoxynucleotides
      • Colours: ddTTP ddGTP ddATP ddCTP
      • These prevent further additions (terminators)
  • Dideoxynucleotides ddNTPs have no 3’ OH, so when added they cannot form the phosphodiester bond required to add the next nucleotide
  • DNA Sequencing Reaction
    • The reaction is boiled to make all the DNA single stranded and then the reaction is resolved on a long polyacrylamide or capillary gel in a DNA sequencer
  • Electropherogram of sequencing gel
  • Decoding DNA sequence data
  • Genotyping Genotyping includes a variety of techniques that are used to identify the primary localization and mapping of genes implicated in human diseases.
      • Polymorphisms (different forms of a gene) may be present in coding and non-coding regions of a gene.
      • Polymorphisms may influence gene regulation in response to nutrients
  • Primer Extension Theory SNP Analysis - primer extension theory
  • SNP Analysis