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In situ hybridization methods and techniques course slides Pat Heslop-Harrison

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Methods and techniques for chromosomal in situ hybridization and molecular cytogenetics. Fixations, chromosomes preparation, mostly using plant chromosomes, hybridiziation mixtures, stringency calculations and fluorescent microscopy.Trude Schwarzacher and Pat Heslop-Harrison

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In situ hybridization methods and techniques course slides Pat Heslop-Harrison

  1. 1. Practical introduction to Molecular Cytogenetics Trude Schwarzacher and Pat Heslop-Harrison www.molcyt.com UserID/PW ‘visitor’ phh4@le.ac.uk @pathh1 Twitter/Youtube/Slideshare 12-14 November 2014
  2. 2. Molecular cytogenetics  Localizes DNA sequences along chromosomes  Answers questions about genome organization and rearrangements  PRACTICAL:  Chromosome preparation and staining  In situ hybridization
  3. 3. Molecular Cytogenetics  Schwarzacher T, Heslop- Harrison JS. 2000. Practical in situ hybridization. Oxford: Bios. 203+xii pp.  "Molecular cytogenetics and the methods of in situ hybridization have revolutionized our understanding of the structure, function, organization, and evolution of genes and the genome..." 
  4. 4. Molecular cytogenetics and Chromosome preparation  Collecting dividing material with chromosomes  Fixation of material  Probe labelling  Chromosome preparation  In situ hybridization  Pretreatment/Probe mix/Denaturation/Hybridization  Washing and hybridization detection  Visualization
  5. 5. Robertsonian Fusion of 1 and 29 to give 2n=58 or 59 Heterozygous rob(1;29) example in Portuguese cattle Barrosa Chaves et al. Chromosome Research
  6. 6. BtSatI BtSatIV FISH on cattle (Brakman) chromosomes Gaspar, Hughes, Chaves and Schwarzacher 2014
  7. 7. Satellite I and II collocalize, Satellite IV has separate arrays BtSatII BtSatI BtSatII BtSatIV Gaspar and Schwarzacher 2014
  8. 8. Molecular Cytogenetics  Schwarzacher T, Heslop-Harrison JS. 2000. Practical in situ hybridization. Oxford: Bios. 203+xii pp.  "Molecular cytogenetics and the methods of in situ hybridization have revolutionized our understanding of the structure, function, organization, and evolution of genes and the genome..."  Review on Amazon: “Next best thing to a course in the authors’ laboratory”!
  9. 9. Thursday am  Laboratory notebook  The metaphase preparation  The hybridization mixture  Denaturation  Control of stringency
  10. 10. From Experiment to Report or Publication  What goes into a publication?  How it is written?  What happens to it after you have completed it?  How do you get a manuscript published?  but first … 11 13/11/2014
  11. 11. Your Laboratory Notebook  This is the document that might end up in court!  Inventorship  Disputed results  Malpractice  Safety “Keeping a laboratory notebook” in Google 12 13/11/2014
  12. 12. Keeping A Laboratory Notebook  A factual account of the work carried out  Title, short introduction and rationale for the experiment (maybe including references to protocols, and key changes you are make to protocols)  Reporting exactly what you used  Reporting exactly what you did  Written at the time of the experiment  Includes pictures/printouts pasted in, or cross-references to (archived) computer files, films, photographs etc.  Brief discussion of results and perhaps what you do with them  Each page numbered, dated, ideally signed and counter-signed
  13. 13. Assessment: A Laboratory Notebook  A factual account of the work carried out  Reporting exactly what you used  Reporting exactly what you did  Written at the time of the experiment  Numbered pages with name on each page  PLUS TO COMPLETE IN THE FOLLOWING WEEK:  Title, short introduction and rationale for the experiment (including references to protocols, and any changes you make to protocols)  Pictures / printouts and other Results  Brief Discussion of results: what they show and the implications  References  Numbered pages with name on each page
  14. 14. Collection of material  Material: needs to divide  root tips  young seedlings  newly grown roots at the edge of plant pots  hydroponic culture  flower buds, anthers, carpels  leaf or apical meristems  Metaphase arresting agents  Colchicine  Hydroxyquinoline  2mM, 30min-2hours at growing temp, 0-2hours at 4oC  Ice water  Herbicides  Fixation: Ethanol:acetic acid 3:1 (VERY fresh!)  Normally half of batches of fixations are not good enough!
  15. 15. Plant chromosome preparation  Rinse fixation in enzyme buffer  Enzyme digestions  Pectinase  Cellulase  37 °C for 20 min to 3 hours  Transfer to enzyme buffer  Continues to soften (can leave 4 °C overnight)  Transfer to 45 or 60% acetic acid  Under the stereomicroscope  Dissect material  Make a single cell layer/suspension  Cover with a coverslip  Disperse chromosomes by tapping with an needle root tips  Squash under a filter paper with thumb  Put on dry ice/liquid nitrogen and remove the coverslip  Air dry
  16. 16. Review of Chromosome Preparation  Fixation  Digestions  Softness can be variable: ‘possible to handle digested roots with care using forceps’ is reasonable aim  18x18 mm coverslips  Best preparations are at edge!  Minimal cell clumps left on slide  Stop coverslip lying flat  Bind excessive probe  Scratches under slide to show cell area
  17. 17. Review of Chromosome Preparation  It’s and ART but experience counts  Roots and slides  Roots: accumulate metaphases  Cleanliness – no fixative near plants, no shocks  Slides: Not all makes ‘work’ (Chromic acid wash?)  Fixation  Use 3:1 fixative less than 30 min old, replace after 2 hours  …
  18. 18.  Triticale roots cv. ‘Lamberto’ (L)  1B-1R wheat cv. ‘Relay’ roots (R)  Sheep chromosome suspension
  19. 19. Chromosomes all to same genome size scale – 2n=10 150Mbp; 2n=46 3,000Mbp; 2n=24 24,000 Mbp
  20. 20. Somatic metaphase chromosomes Arabidospsis Human Pine Centromere Attachment site for microtubuli Telomere End of the chromosomes
  21. 21. In situ hybridization Pretreatment of chromosome preparations  RNAse treatment  Protease treatment - permeabilization  Pepsin  Proteinase K  Acetylation  Refixation – prevent loss of material  4% paraformaldehyde
  22. 22. In situ hybridization  Fixation of material  Chromosome preparation  Probe labelling  In situ hybridization  Pretreatment of chromosome preparations  Probe mix  Denaturation  Hybridization  Detection  Fluorescent DNA staining  Visualization
  23. 23. Denature chromosomes Make single stranded Let reanneal hybridize
  24. 24. Techniques  The most important reagent: WATER Bottled drinking water for seed germination Purchased molecular biology water dissolving DNA, reactions <1 ml Water purification/distillation
  25. 25. SINE A2/tA is part of Satellite IV and hybridizes to euchromatin and centromeric heterochromatin
  26. 26. Banana: 2n=3x=33
  27. 27. Chromosomal Markers  Total genomic DNA  Genomes in hybrids  Polyploidy is critical part of plant evolution  Chromosomes in backcrosses  Widely used for gene transfer  Chromosomal segments  Repetitive DNA sequences
  28. 28. In situ hybridization Probe mixture  Formamide: 50%  SSC: 2x  Dextran sulphate: 20%  Detergent: 0.1% SDS  EDTA: 1.25 mM  Salmon Sperm DNA: 1-5 μg/slide  Probe DNA: 25-100ng/slide  Blocking DNA: 2-100x probe DNA
  29. 29. In situ hybridization Stringency Amount of mismatches that are allowed
  30. 30. Control of hybridization stringency  “This chapter should be compulsory reading for all PhD students in molecular biology … and their supervisors too” Review of Practical in situ hybridization, Heredity
  31. 31. In situ hybridization Melting temperature of DNA Tm = 0.41(%GC of probe) + 16.6 log (molarity of monovalent cations) – 500/(probe fragment length) – 0.61 (% formamide) + 81.5°C  Temperature: high  Formamide: high  Monovalent cations: low  Na+ in SSC (saline sodium citrate)  Probe lengths: short  Mismatch: many
  32. 32. Review of stringency control  Melting Temperature (DS  SS) of DNA = Tm  Tm= 0.41*(%GC) + 16.6 log[Na+] - 0.61(%formamide) - 500/(probe length) + 81.5  % mismatch allowed changes by 1%/°C  See tables in Chapter 7 of in situ book
  33. 33. In situ hybridization Stringency Amount of mismatch that is allowed  Stringency = 100 – Mf (Tm – Ta)  Mf for probes 150bp = 1  Change of 1oC = 1%  Stringency of 80% allows 20% mismatch
  34. 34. In situ hybridization Probe mixture  Formamide: 50%  SSC: 2x  Dextran sulphate: 20%  Detergent: 0.1% SDS  EDTA: 1.25 mM  Salmon Sperm DNA: 1-5 μg/slide  Probe DNA: 25-100ng/slide  Blocking DNA: 2-100x probe DNA
  35. 35. In situ hybridization  Denaturation  70-80oC, 5-10 mins  Slow cooling down  Hybridization  37oC, 12-36hours
  36. 36. In situ hybridization Stringency control By hybridization mixture By washes  In situ hybridization  Day 2: Washing, stringent washing and detection  Fluorescent DNA staining  Visualization
  37. 37. From Chromosome to Nucleus Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com
  38. 38. Cell fusion hybrid of two 4x tetraploid tobacco species Nicotiana hybrid 4x + 4x cell fusions Each of 4 chromosome sets has distinctive repetitive DNA when probed with genomic DNA Four genomes differentially labelled Patel, Patel et al Badakshi, Ann Bot 2011 HH, Davey et al 2011
  39. 39. Size and location of chromosome regions from radish (Raphanus sativus) carrying the fertility restorer Rfk1 gene and transfer to spring turnip rape (Brassica rapa) DAPI metaphase blue Radish genomic red (2 radish chromosomes) far-red 45S rDNA Rfk1 carrying BAC green labels sites on radish and homoeologous pair in Brassica Tarja Niemelä, Seppänen, Badakshi, Rokka HH Chromosome Research 2012
  40. 40. BACs from different species have different repeat distributions – and hence different patterns of hybridization
  41. 41. Satellite DNA probe green
  42. 42. Differences between genomes Major differences in the nature and amount of repetitive DNA • 45S rDNA • dpTa1 tandem repeat
  43. 43. Genes!
  44. 44. Technology and Methods  DNA in situ hybridization  Localizes sequences to chromosomes – organization and variation  Southern hybridization  Data about sequence organization and variation  PCR-based analyses  Sequence analysis  ‘In silico’ results
  45. 45.  .
  46. 46. Fluorescent chromosome staining stains interact with the DNA  AT-rich  DAPI 4’,6-diamidino-2-phenylindole  Hoechst 33258  GC-rich  Chromomycin A3  No bias  Ethidium bromide  Propidium iodide
  47. 47. Fluorescent chromosome staining Fluorophores or fluorescent dyes  Are excited with light of a given wavelengths  Use the energy of the light  Emit light of higher wavelength = less energy  Have a definite life = fading  Description/definition of fluorophores  Excitation maxima  Emission maxima
  48. 48. Fluorochrome Excitation Emission Fluorophores Amino-methyl coumarin (AMCA)** 399nm 445nm Cyanine 2 (Cy2) 489nm 506nm Alexa 488 490nm 520nm Fluorescein isothiocyanate (FITC)** 495nm 523nm Alexa 532 525nm 550nm Tetramethylrhodamine isothiocyanate (TRITC) 550nm 570nm Cyanine 3 (Cy3) 550nm 570nm Alexa 546 555nm 570nm Rhodamine B** 560nm 580nm Texas red** 595nm 610nm Alexa 594 590nm 615nm BODIPY 650/665 650nm 670nm Cyanine 5 (Cy5) 649nm 670nm Cyanine 7 (Cy7) 743nm 767nm DNA stains 4’,6-Diamidino-2-phenylindole (DAPI) 358nm 461nm Hoechst 33258 (bis-benzimide) 352nm 461nm Chromomycin A3 430nm 570nm Ethidium bromide 518nm 615nm Propidium iodide 535nm 617nm
  49. 49. Probes  Clones  Plasmids  BACs  Synthetic Oligos  PCR products  Genomic DNA
  50. 50. Nick translation labelling
  51. 51. Random primer
  52. 52. Repetitive Sequences in the Genome
  53. 53. 2.5 Genomics – The genome and retroelements Retroelements Sequences which amplify through an RNA intermediate 30% to 50% of all the DNA!
  54. 54. Retroelement Markers LTR Retrotransposon LTR Random sites Insertion SSAP IRAP – InterRetroelement PCR LTR Retrotransposon LTR LTR Retrotransposon LTR LTR Retrotransposon LTR REMAP – Retroelement Microsat Amplified Polymorphisms LTR Retrotransposon LTR Simple sequence repeat
  55. 55. End labelling
  56. 56. PCR Labelling
  57. 57. Let reanneal hybridize
  58. 58. Practical introduction to Molecular Cytogenetics Trude Schwarzacher and Pat Heslop-Harrison www.molcyt.com UserID/PW ‘visitor’ phh4@le.ac.uk @pathh1 Twitter/Youtube/Slideshare 12-14 November 2014

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