in situ hybridization methods and techniques course slides pat heslop-harrison

Practical introduction to

Molecular Cytogenetics

Trude Schwarzacher and Pat Heslop-Harrison

www.molcyt.comUserID/PW ‘visitor’

phh4@le.ac.uk

@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

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

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’

phh4@le.ac.uk

@pathh1 Twitter/Youtube/Slideshare

12-14 November 2014