M sc2

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  • M sc2

    1. 1. Molecular Approaches to Nutrition <ul><li>Molecular Biology 2 </li></ul><ul><li>Principles and Methods Dr. Janice Drew </li></ul>
    2. 2. Principles and Methods <ul><li>Purification and handling of DNA/RNA </li></ul><ul><li>Gel Electrophoresis </li></ul><ul><li>Nucleic Acid Hybridisation </li></ul><ul><li>Cutting and rejoining DNA </li></ul><ul><li>Methods of introducing DNA into cells </li></ul><ul><li>PCR </li></ul><ul><li>DNA sequencing </li></ul><ul><li>Sequence interpretation </li></ul>
    3. 3. Handling of DNA/RNA <ul><li>DNases and RNases </li></ul><ul><li>Glass and plasticware </li></ul><ul><li>Solutions </li></ul>
    4. 4. Extraction of DNA/RNA <ul><li>DNA extraction </li></ul><ul><li>Alkaline lysis </li></ul><ul><li>Neutralisation </li></ul><ul><li>Precipitation of proteins and cell debris </li></ul><ul><li>Precipitation or elution using spin column </li></ul><ul><li>RNA extraction </li></ul><ul><li>Lysis incorporating instantaneous inactivation of RNases </li></ul><ul><li>Separation of contaminating DNA </li></ul><ul><li>Precipitation or elution using spin column </li></ul>
    5. 5. Quantitation and analysis of DNA/RNA <ul><li>Spectrometric determination at 260nm </li></ul><ul><li>Gel Electrophoresis </li></ul><ul><li>Agilent technology </li></ul>
    6. 6. Gel Electrophoresis <ul><li>Nucleic acids are negatively charged </li></ul><ul><ul><li>PO 4 - groups </li></ul></ul><ul><li>Electrophoresis resolves by size </li></ul><ul><li>Agarose is the usual gel matrix </li></ul><ul><li>Ethidium bromide/SYBR green ‘stains’ DNA & RNA </li></ul><ul><ul><li>Fluorescent colour under UV illumination </li></ul></ul>
    7. 7. 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
    8. 8. Agarose Gel Image
    9. 9. Agilent Technology
    10. 10. Electropherogram showing Agilent analysis of total RNA 18S 28S Times (seconds) Fluorescence
    11. 11. Hybridisation - Identification of DNA/RNA <ul><li>Agarose gel electrophoresis separates nucleic acids on the basis of size - does not identify DNA/RNA fragments </li></ul><ul><li>Nucleic acid probes are used to identify specific DNA/RNA sequences in a gel </li></ul><ul><li>Probe is a known nucleic acid sequence </li></ul><ul><li>Relies on the principle of base pairing - complementary DNA/RNA sequences stick (hybridise) together </li></ul>
    12. 12. Hybridisation - Identification of DNA/RNA <ul><li>Many molecular biology procedures to identify specific DNA/RNA sequences use this principle - </li></ul><ul><li>Southern (DNA) or Northern (RNA) blotting </li></ul><ul><li>In situ hybridisation </li></ul><ul><li>Microarrays </li></ul><ul><li>Antisense technologies </li></ul>
    13. 13. Probe Production <ul><li>Synthesise a known fragment </li></ul><ul><li>OR </li></ul><ul><li>Purify a known fragment of DNA </li></ul><ul><ul><li>Restriction enzyme digestion </li></ul></ul><ul><li>Heat denature to give single strands </li></ul><ul><li>Add primers, DNA polymerase and radioactive/colour labelled nucleotides </li></ul><ul><ul><li>Make a radioactive/ colour labelled complementary strand </li></ul></ul><ul><li>Denature to give single strands </li></ul>
    14. 14. 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
    15. 15. Restriction Endonucleases <ul><li>Restriction endonucleases cut DNA </li></ul><ul><li>Present in bacteria </li></ul><ul><li>Cut at sequence specific sites </li></ul><ul><ul><li>Usually 4 or 6 base pairs long </li></ul></ul><ul><li>Bacteria use them to destroy ‘foreign’ DNA </li></ul><ul><ul><li>Bacteria protect their own DNA against self-cutting by special methylation of their DNA </li></ul></ul><ul><li>Restriction enzymes can be purified and are used in genetic engineering studies </li></ul>
    16. 16. Restriction Endonucleases <ul><li>Example Restriction enzymes </li></ul><ul><ul><li>Eco R I ( E . co li R estriction Endonuclease I ) </li></ul></ul><ul><ul><li>Stu I ( S treptomyces tu bercidicus I) </li></ul></ul>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
    17. 17. Molecular Scissors and Glue <ul><li>There are 100’s of restriction enzymes, each one with a different recognition site </li></ul><ul><ul><li>These enzymes are ‘molecular scissors’ and can be used to specifically cut long DNA strands into smaller pieces </li></ul></ul><ul><li>The T4 virus, which infects E. coli , has an enzyme, T4 DNA ligase, which can form a phosphodiester bonds between DNA molecules </li></ul><ul><ul><li>Purified T4 DNA ligase can be used as ‘molecular glue’ to join pieces of DNA. This enzyme is widely used for DNA cloning </li></ul></ul>
    18. 18. 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
    19. 19. Methods of introducing DNA into cells <ul><li>Plasmids </li></ul><ul><li>Viruses </li></ul><ul><li>DNA and RNA viruses </li></ul><ul><li>Phage vectors </li></ul>
    20. 20. Cloning DNA into Plasmids <ul><li>Bacteria have a circular DNA genome </li></ul><ul><ul><li>5 to 10 million base pairs (bp) in size </li></ul></ul><ul><li>Many bacteria also contain plasmids </li></ul><ul><ul><li>Small circular DNA molecules, ~3,000 to 50,000 bp </li></ul></ul><ul><ul><li>Note : The bacterial genome is not a plasmid </li></ul></ul><ul><li>Plasmids contain ‘extra’ genes which are often vital for the survival of the bacterium </li></ul><ul><ul><li>Nutrient metabolism, antibiotic resistance </li></ul></ul><ul><li>Plasmids can be used as vectors in which foreign DNA can be ligated (cloned) </li></ul>
    21. 21. 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 .
    22. 22. 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)
    23. 23. DNA and Retroviruses can serve as vehicles for the introduction of new DNA into a cell
    24. 24. DNA / RNA viruses as ‘vehicles’ Chromosomal DNA Viral DNA Integration into genome gene x Gene Therapy and Transgenics
    25. 25. Polymerase Chain Reaction (PCR) <ul><li>PCR generates multiple copies of DNA </li></ul><ul><ul><li>Heat resistant DNA polymerase used to copy a section of DNA e.g Taq </li></ul></ul><ul><li>Very efficient copying </li></ul><ul><ul><li>Billions of copies from a single ‘template’ DNA </li></ul></ul><ul><li>Small volume / quick analysis </li></ul>
    26. 26. Polymerase Chain Reaction (PCR) <ul><li>Entire reaction performed in single tube </li></ul><ul><ul><li>10 to 50 μ l volume </li></ul></ul><ul><li>Reaction contains </li></ul><ul><ul><li>Template DNA, heat resistant DNA polymerase, a pair of specific DNA primers (in excess over the template), nucleotide bases, appropriate reaction buffer </li></ul></ul><ul><li>Reaction is repeatedly cycled through 3 temperatures (x30) </li></ul><ul><ul><li>95 ° C (makes DNA single stranded) </li></ul></ul><ul><ul><li>~55 - 60 ° C (primers anneal to template DNA) </li></ul></ul><ul><ul><li>72 ° C DNA polymerase copies DNA, starting from the primers </li></ul></ul>
    27. 27. A Thermocycler This thermocycler can accept 1500 reactions at a time, and complete them in 2 to 4 hours.
    28. 28. 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
    29. 29. DNA Sequencing <ul><li>A specific primer binds to denatured DNA </li></ul><ul><li>Heat resistant DNA polymerase extends a new strand from this primer </li></ul><ul><li>Complementary nucleotides are added as appropriate </li></ul><ul><li>In the reaction are small quantities of coloured dideoxynucleotides </li></ul><ul><ul><li>Colours: ddTTP ddGTP ddATP ddCTP </li></ul></ul><ul><ul><li>These prevent further additions (terminators) </li></ul></ul>
    30. 30. Dideoxynucleotides ddNTPs have no 3’ OH, so when added they cannot form the phosphodiester bond required to add the next nucleotide
    31. 31. DNA Sequencing Reaction <ul><li>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 </li></ul>
    32. 32. Electropherogram of sequencing gel
    33. 33. Decoding DNA sequence data
    34. 34. Genotyping Genotyping includes a variety of techniques that are used to identify the primary localization and mapping of genes implicated in human diseases. <ul><ul><li>Polymorphisms (different forms of a gene) may be present in coding and non-coding regions of a gene. </li></ul></ul><ul><ul><li>Polymorphisms may influence gene regulation in response to nutrients </li></ul></ul>
    35. 35. Primer Extension Theory SNP Analysis - primer extension theory
    36. 36. SNP Analysis

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