in situ hybridization methods and techniques course slides pat heslop-harrison
DESCRIPTION
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-HarrisonTRANSCRIPT
Practical introduction to
Molecular Cytogenetics
Trude Schwarzacher and Pat Heslop-Harrison
www.molcyt.comUserID/PW ‘visitor’
@pathh1 Twitter/Youtube/Slideshare
12-14 November 2014
Molecular cytogenetics
Localizes DNA sequences along
chromosomes
Answers questions about genome
organization and rearrangements
PRACTICAL:
Chromosome preparation and staining
In situ hybridization
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..."
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
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
BtSatI BtSatIV
Gaspar, Hughes, Chaves and Schwarzacher 2014
FISH on cattle
(Brakman)
chromosomes
Satellite I and II collocalize,
Satellite IV has separate arrays
BtSatII BtSatI BtSatII BtSatIVGaspar and Schwarzacher 2014
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”!
Thursday am
Laboratory notebook
The metaphase preparation
The hybridization mixture
Denaturation
Control of stringency
13/11/201411
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 …
13/11/201412
Your Laboratory Notebook
This is the document that might end up in
court!
Inventorship
Disputed results
Malpractice
Safety
“Keeping a laboratory notebook” in Google
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
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
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!
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
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
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
…
Triticale roots cv. ‘Lamberto’ (L)
1B-1R wheat cv. ‘Relay’ roots (R)
Sheep chromosome suspension
Chromosomes all to same genome size scale – 2n=10 150Mbp; 2n=46 3,000Mbp; 2n=24 24,000 Mbp
CentromereAttachment site for microtubuli Telomere
End of the chromosomes
Somatic metaphase chromosomes
Arabidospsis Human Pine
In situ hybridization Pretreatment of chromosome preparations
RNAse treatment
Protease treatment - permeabilization
Pepsin
Proteinase K
Acetylation
Refixation – prevent loss of material
4% paraformaldehyde
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
Denature chromosomes
Make single stranded
Let reanneal
hybridize
Techniques
The most important reagent:
WATER
Bottled drinking water for
seed germination
Purchased molecular biology water
dissolving DNA, reactions <1 ml
Water purification/distillation
SINE A2/tA is part of Satellite IV and hybridizes to
euchromatin and centromeric heterochromatin
Banana: 2n=3x=33
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
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
In situ hybridization Stringency
Amount of mismatches that are allowed
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
In situ hybridization Melting temperature of DNA
Tm = 0.41(%GC of probe) + 16.6 log (molarity ofmonovalent 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
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
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
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
In situ hybridization
Denaturation
70-80oC, 5-10 mins
Slow cooling down
Hybridization
37oC, 12-36hours
In situ hybridization
Stringency control
By hybridization mixture
By washes
In situ hybridization
Day 2: Washing, stringent washing and detection
Fluorescent DNA staining
Visualization
Nicotianahybrid
4x + 4x
cell fusions
Each of 4
chromosome
sets has
distinctive
repetitive
DNA when
probed with
genomic DNA
Patel et al
Ann Bot 2011
Cell fusion
hybrid of two
4x tetraploid
tobacco
species
Four
genomes
differentially
labelled
Patel,
Badakshi,
HH, Davey et
al 2011
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
BACs from different
species have different
repeat distributions – and
hence different patterns
of hybridization
Satellite DNA probe green
• 45S rDNA
Differences between genomes
Major differences in the nature and amount of
repetitive DNA
• dpTa1 tandem repeat
Genes!
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
.
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
Fluorescent chromosome stainingFluorophores 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
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
Probes
Clones
Plasmids
BACs
Synthetic Oligos
PCR products
Genomic DNA
Nick translation labelling
Random primer
Repetitive Sequences in the
Genome
RetroelementsSequences which amplify through an RNA intermediate
30% to 50% of all the DNA!
2.5 Genomics – The genome and retroelements
Retroelement Markers
Retrotransposon LTRLTR
Retrotransposon LTRLTR
RetrotransposonLTR LTR
Retrotransposon LTRLTR Simple sequence repeat
Retrotransposon LTRLTR
Random sites
Insertion
SSAP
IRAP – InterRetroelement PCR
REMAP – Retroelement Microsat Amplified Polymorphisms
End labelling
PCR Labelling
Let reanneal
hybridize
Practical introduction to
Molecular Cytogenetics
Trude Schwarzacher and Pat Heslop-Harrison
www.molcyt.comUserID/PW ‘visitor’
@pathh1 Twitter/Youtube/Slideshare
12-14 November 2014