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

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

3. Molecular cytogenetics
 Localizes DNA sequences along
chromosomes
 Answers questions about genome
organization and rearrangements
 PRACTICAL:
 Chromosome preparation and staining
 In situ hybridization

4. 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..."


5. 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

6. 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

7. BtSatI BtSatIV
FISH on cattle
(Brakman)
chromosomes
Gaspar, Hughes, Chaves and Schwarzacher 2014

8. Satellite I and II collocalize,
Satellite IV has separate arrays
BtSatII BtSatI BtSatII BtSatIV
Gaspar and Schwarzacher 2014

9. 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”!

10. Thursday am
 Laboratory notebook
 The metaphase preparation
 The hybridization mixture
 Denaturation
 Control of stringency

11. 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

12. 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

13. 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

14. 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

16. 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!

19. 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

20. 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

21. 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
 …

22.  Triticale roots cv. ‘Lamberto’ (L)
 1B-1R wheat cv. ‘Relay’ roots (R)
 Sheep chromosome suspension

23. Chromosomes all to same genome size scale – 2n=10 150Mbp; 2n=46 3,000Mbp; 2n=24 24,000 Mbp

24. Somatic metaphase chromosomes
Arabidospsis Human Pine
Centromere
Attachment site for microtubuli Telomere
End of the chromosomes

27. In situ hybridization
Pretreatment of chromosome preparations
 RNAse treatment
 Protease treatment - permeabilization
 Pepsin
 Proteinase K
 Acetylation
 Refixation – prevent loss of material
 4% paraformaldehyde

28. 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

29. Denature chromosomes
Make single stranded
Let reanneal
hybridize

30. Techniques
 The most important reagent:
WATER
Bottled drinking water for
seed germination
Purchased molecular biology water
dissolving DNA, reactions <1 ml
Water purification/distillation

31. SINE A2/tA is part of Satellite IV and hybridizes to
euchromatin and centromeric heterochromatin

33. Banana: 2n=3x=33

34. 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

36. 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

37. In situ hybridization
Stringency
Amount of mismatches that are allowed

38. 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

39. 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

40. 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

41. 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

42. 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

43. In situ hybridization
 Denaturation
 70-80oC, 5-10 mins
 Slow cooling down
 Hybridization
 37oC, 12-36hours

44. In situ hybridization
Stringency control
By hybridization mixture
By washes
 In situ hybridization
 Day 2: Washing, stringent washing and detection
 Fluorescent DNA staining
 Visualization

46. From Chromosome to Nucleus
Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

47. 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

48. 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

49. BACs from different
species have different
repeat distributions – and
hence different patterns
of hybridization

50. Satellite DNA probe green

52. Differences between genomes
Major differences in the nature and amount of
repetitive DNA
• 45S rDNA
• dpTa1 tandem repeat

53. Genes!

55. 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

57.  .

58. 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

59. 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

60. 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

61. Probes
 Clones
 Plasmids
 BACs
 Synthetic Oligos
 PCR products
 Genomic DNA

63. Nick translation labelling

64. Random primer

65. Repetitive Sequences in the
Genome

68. 2.5 Genomics – The genome and retroelements
Retroelements
Sequences which amplify through an RNA intermediate
30% to 50% of all the DNA!

69. 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

70. End labelling

71. PCR Labelling

72. Let reanneal
hybridize

73. 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