Human Genome Project Resources for
The announcement in April 2003 that the Human
Genome Project was complete and was available
publicly through intuitive genome browsers (e.g.,
see http://www.ensembl.org/Homo_sapiens/ and
experimental genome analysis. More precisely
the study of chromosome rearrangements has
been facilitated by the availability of easily accessible,
ordered, fully sequenced tiling path clones covering
the entire human genome. This resource enables chromosome
breakpoints to be mapped more rapidly and
with increased confidence.
Conventionally a chromosome rearrangement has
been mapped by walking along a chromosome
sequentially hybridising fluorescently labelled DNA
clones (FISH) until the signal is no longer retained on
one derivative chromosome but is seen on the reciprocal
derivative chromosome. The genomic position of
the breakpoint is then delineated by these two clones
and the spanning clone can be found by FISHing
clones at a higher resolution (McMullan et al.
The genome annotation also now easily available on
web browsers (e.g., http://vega.sanger.ac.uk/
Homo_sapiens/) provides an additional level of information
regarding the gene content of a genomic region
of interest. Both known and predicted genes are
detailed along with additional information, including
repeat content, GC content, mouse homology, and
many additional features. This information, together
with the identification of breakpoint spanning clones
linked directly to these databases, allows candidate
genes involved in the phenotype of a patient to be
|FIGURE 1 Partial flow karyotype of
Clones sequenced as part of the Human Genome
Project are available readily to international
researchers for minimal cost (e.g., BACPAC resources:
http://bacpac.chori.org/, Invitrogen: http://clones.
invitrogen.com/, and the Resources for Molecular
Cytogenetics, University of Bari: http://www.biologia.
uniba.it/rmc/). The sequence obtained from these
clones is freely accessible (e.g., http://www.ncbi.nlm.
Homo_sapiens/, and http://genome.ucsc.edu/cgi-bin/hgGateway).
The availability of such a comprehensive set of
clones has not only greatly facilitated conventional
approaches to breakpoint mapping studies but has
also enabled the development of genome-wide, large
insert clone DNA microarrays. The selection, DNA
extraction, and labelling of clones for multiple sequential
DNA clone in situ
required to map breakpoints are often time-consuming
processes. However, the clones can be used as ordered
targets themselves for breakpoint mapping after
gridded assembly on a DNA microarray (Fiegler et al.
2003a). DNA microarrays have been recently developed
mostly with the aim of studying genome imbalance
in tumour DNA samples (Solinas-Toldo et al.
1997). We have exploited this resource to study
structural chromosome rearrangements, including
rearrangements with no genomic imbalance. This type
of analysis, termed array painting (Fiegler et al.
involves isolating derivative chromosomes away from
other chromosome homologues by flow sorting
(Carter, 1994) or microdissection prior to hybridisation
onto the DNA microarrays.
|FIGURE 2 Basic principls of mapping
translocation breakpoints by array painting.
The flow sorting strategy (see Fig. 1) enables large
quantities of derivative chromosomes to be isolated
before they are fluorescently labelled and hybridised onto a target DNA microarray. Alternatively, smaller
quantities (~500) of pure derivative chromosomes
can be amplified using whole genome amplification.
(WGA) procedures, such as DOP-PCR (Telenius et al.
1992; Fiegler et al.
, 2003a), Repli-G (Qiagen, 59043),
or GenomiPhi (Amersham Biosciences, 25-6600-01)
before they are fluorescently labelled and hybridised
onto the array. Analysis of this type of hybridisation
will then identify the components of each derivative
chromosome according to the fluorescence
intensities exhibited by the clones on the array (see
The limit of array painting as a tool for mapping
chromosome breakpoints is defined by the resolution
of the clones gridded. At present we use a 1-Mb array
to define the breakpoints to within a megabase (see
Fig. 3) and then use
conventional FISH methods (see
article by Leversha) to refine the breakpoints and find
the spanning clones. Clones that span the breakpoint
show intermediate ratios on the array (see Fig. 2). Statistically, 1 in every 10 breakpoints will have spanning
clones identified directly on the 1-Mb resolution
This technique has proven to be invaluable in the
detailed study of chromosome rearrangements. When
used in conjunction with array CGH, it can give an
insight into rearrangements that might have been
undetected by G-banding analysis. Increased array
resolution, e.g., a tiling path array, in the study of
simple chromosome rearrangements would identify
spanning clones within a single experiment.
II. MATERIALS AND
A. Purification of Flow-Sorted DNA
|FIGURE 3 Array painting profiles for the derivative chromosomes of t(17;22): (A)1-Mb array chromosome
17 plot and (B)1-Mb array chromosome 22 plot.
-Lauroylsarcosine sodium salt (Sigma, L5125)
Proteinase K (Invitrogen, 25530-015)
Phenylmethylsulfonyl fluoride (PMSF) (Sigma, P7626)
Pellet Paint nonfluorescent coprecipitant (Novagen,
B. Random Labeling of DNA for Array
BioPrime DNA labeling kit (Invitrogen, 18094-011)
contains 2.5× random primers solution, sterile
water, Klenow fragment, and stop buffer
dNTPs (Amersham Biosciences, 27-2035-02)
Cyanine 3-dCTP (Perkin Elmer, NEL576001EA)
Cyanine 5-dCTP (Perkin Elmer, NEL577001EA)
Microspin G50 columns (Amersham Biosciences, 27-
C. Array Hybridization
Herring sperm DNA (Sigma, D7290)
Human Cot-1 DNA (Roche, 1581074)
Deionised formamide for hybridisation buffer (Sigma,
Dextran sulphate sodium salt (Amersham Biosciences,
HPLC water (VWR International, 152736D)
Rubber cement (Weldtite, 02002)
Formamide (Sigma, 47670)
Yeast tRNA (Invitrogen, 15401-029)
Tween 20 (VWR International, 663684B)
Hellendahl jars (RA Lamb, E95)
Glass troughs (RA Lamb, E106 + E98)
Parafilm (Sigma, P7793)
Shake "n" stack hybridisation oven (Thermo Hybaid,
A. Purification of Flow-Sorted DNA
- 0.25 M EDTA/10% sodium lauroyl sarcosine: For 10 ml,
dissolve 1 g N-lauroylsarcosine in 5 ml filtered water
and add 5ml 0.5M EDTA (pH 8.0). Store in 1ml
aliquots at 4°C.
- Profeinase K: Resuspend 100mg in 5 ml sterile water
(final concentration of 20mg/ml). Store 50µl
aliquots at -20°C. Do not freeze thaw.
- PMSF: Resuspend 250mg in 12.5ml 96% ethanol
(final concentration of 20mg/ml). Store at 4°C up to
a maximum of 6 months.
- 5M NaCl: Dissolve 29.22g NaCl (MW 58.44) in
100ml sterile water. Autoclave and store at room
- Pellet Paint: Store as 20µl aliquots at -20°C. Avoid
repeated freeze thawing. Working tube can be kept
- Absolute ethanol: Store in a sterile 50ml Falcon tube
- 70% ethanol: Store in a sterile 50ml Falcon tube at
- Sterile T1.0E (pH 8.0): For 100ml, mix 1ml 1M
Tris-HCl (pH 8.0), 1ml 0.1M EDTA (pH 8.0), and
98 ml water. Autoclave and store at room temperature.
Handle tubes containing flow-sorted material very
Flow-sorted chromosomes come in batches of
approximately 250,000 per 1.5ml microfuge tube,
sorted into chromosome sheath buffer [10mM
Tris-HCl (pH 8.0), 1mM
NaCl, 0.5 mM
Na azide]. Sort 250,000 chromosomes into a volume of
approximately 300µl. Amounts of reagents will vary
according to the quantity of sorted sample.
B. Random Labeling of DNA for Array
- Add 30µl 0.25M EDTA/10% sodium lauroyl sarcosine
and 3 µl proteinase K. Incubate overnight at 42°C.
- Dilute stock PMSF to 4mg/ml in 96% ethanol.
Add 3.33 µl to sorted DNA tubes (40 µg/ml final). Incubate
for 40min at room temperature.
- Add 13.44 µl 5M NaCl (final 0.3M including
sheath buffer), 2µl Pellet Paint, and 770µl absolute
ethanol. Mix gently by inversion. Precipitate overnight
at -20°C or at -70°C for 30min. (Tubes can be stored
at this stage at -20°C if necessary.)
- Pellet DNA in microfuge at full speed (11,000g)
at room temperature for 15min. The hinge of the
Eppendorf tube should face outwards so that the pellet
is easily located.
- Remove most of the supernatant with a P1000.
Remove the remainder with P200, avoiding the pellet.
Add 1ml 70% ethanol without disturbing the pellet
and then spin again for 7 min. Remove supernatant as
before. Allow pellets to air dry (about 20min).
- Add 50µl TE directly to the pellet to resuspend.
DNA should separate from the wall of the tube immediately.
Mix by very gentle flicking. Allow DNA to dissolve
for at least 2h.
- 2.5× random primer solution: Found in BioPrime kit.
Store at -20°C and use as supplied.
- Sterile water: Found in BioPrime kit. Store at -20°C and use as supplied.
- 10× dNTP mix: To make 1ml, mix 10µl dCTP (1 mM final), 20 µl dATP (2 mM final), 20 µl dGTP (2 mM final), and 20µl dTTP (2mM final) in 930µl TE
buffer. Store 1ml aliquots at -20°C.
- Cy3-dCTP: Store as stock in the dark at -20°C.
- Cy5-dCTP: Store as stock in the dark at -20°C.
- Klenow fragment: Found in BioPrime kit. Store at
-20°C and use as supplied.
- Stop buffer: Found in BioPrime kit. Store at -20°C and use as supplied.
Tubes containing Cy dyes should be kept in the dark!
1. Labeling Reactions
2. Clean-up of Labeling Reactions
- To 50µl resuspended chromosomes, add 60µl 2.5×
random primers solution and add 20.5µl sterile
water to make up to 130.5µl.
- Denature samples in a hot block for 10min at 100°C and immediately cool on ice.
- Add the following reagents on ice: 15µl 10x dNTP
mix, 1.5µl Cy3- or Cy5-1abelled dCTP, and 3µl
Klenow fragment. Mix thoroughly but gently.
- Incubate the reaction at 37°C overnight.
- Stop the reaction by adding 15µl stop buffer.
Use 3 columns per labelling reaction. Once the
columns have been prepared, they must be used
immediately to avoid the resin drying out.
Sample Application Steps
- Resuspend the resin in the columns by gentle
- Loosen the cap one quarter turn and snap off the
- Place the column in a 1.5 ml screw-capped tube.
- Spin the columns for exactly 1 min at 735g.
C. Array Hybridization
- Place the columns in a new 1.5 ml tube and slowly
apply 55µl of the labelled sample to the centre of
the angled surface of the resin bed, being careful not
to disturb it. Do not allow any of the sample to flow
around the side of the resin bed.
- Spin the columns for 2 min at 735g. Collect the purified
samples in the support tube.
- Discard the columns.
- Combine the three samples.
- Run 5µl on a 1% agarose gel to check for correct
labelling. Ideally, the fragments should range
from 200 to 800bp in size. Tubes containing the
samples should be pink (Cy3) and blue (Cy5) in
colour, indicating incorporation of the fluorescent
- Human Cot-1 DNA: Use as supplied (1 µg/ul). Store
- 3 M NaAc pH 5.2: For 200ml, dissolve 49.2 g NaAc
(anhydrous) in ~140ml H2O, pH to 5.2 with glacial
acetic acid, make up to 200ml, and then autoclave.
Store at room temperature.
- Absolute ethanol: Store in a sterile 50 ml Falcon tube
- Herring sperm DNA: Use as supplied (10mg/ml).
Store at -20°C.
- 40%formamide/2xSSC: To make up 50ml, mix 20ml
formamide, 5 ml 2x SSC, and 25 ml sterile double
distilled water (DDW). Store in a 50 ml Falcon at
- Hybridisation buffer: For 10ml, mix 5ml deionised
formamide, 2 ml 50% dextran sulphate, 100 µl Tris,
pH 7.4, 10µl Tween 20, 1ml 2x SSC, and 1890µl
- 80% ethanol: Store in a sterile 50ml Falcon tube at
- Yeast tRNA: Make up to a final concentration of
100µg/µl by dissolving in autoclaved water and
store as 50-µl aliquots at -20°C.
- 20% formamide/2xSSC: To make up 50ml, use 10ml
formamide, 5 ml 2x SSC, and 35 ml DDW. Store
in a 50ml Falcon at 4°C.
- PBS/O.05% Tween 20: Add 500µl Tween 20 to 1 liter
phosphate-buffered saline (PBS).
- 50% formamide/2xSSC: For 400ml, mix 200ml formamide,
40ml 20xSSC, and 160ml HPLC water.
Incubate at 42°C prior to use.
2. Preparation of Slide
- Prepare 2 × 2ml Eppendorfs.
- Heat the herring sperm DNA on a 70°C hot block
for 5 min before use.
- To tube 1 add 180µl Cy3-labelled DNA, 180µl Cy5-
labelled DNA, 135 µl human Cot-1 DNA, 55 µl 3 M NaAc, pH 5.2, and 1400µl 100% ice-cold ethanol.
- To tube 2 add 80µl herring sperm DNA, 135µl
human Cot-1 DNA, 23µl 3M NaAc, pH 5.2, and
600 µl ice-cold 100% ethanol.
- Mix the tubes gently and precipitate in the dark at
-20°C overnight or at -70°C for 30min.
3. Preparation of Humidity Chambers
- Place the slide over a dummy slide identifying
the array area and apply a rubber cement ring closely
around the grid using a small syringe, taking care not
to go over the grid. Avoid thin threads of the cement
going onto the array as you pull the syringe tip away.
- After the first layer has dried, carefully apply a
second layer over the first one.
- In a fume hood, prepare a humidity chamber
by pipetting a 40% formamide/2xSSC mix onto a
Whatman paper strip until the paper is damp.
- Leave the humidity chamber in the fume hood
ready for use.
- Preheat the hybridisation buffer in a 70°C hot
- Spin the precipitated DNAs for 15 min at 9500g.
- Remove the supernatant and add 500µl 80%
- Respin at 9500g for 5 min.
- Remove the supernatant with a P1000 and then
- Respin at 9500g for 1 min.
- Remove any remaining supernatant with a P10
until the pellet is dry.
- Resuspend the DNAs in 60µl hybridisation
buffer and 6µl yeast tRNA (tube 1) and 140µl hybridisation
buffer (tube 2). This is best done by adding the
hybridisation buffer and leaving the tube for 2-3 min
in a 70°C hot block before resuspending the pellet
using a displacement pipette. Ensure that the DNA is
properly resuspended before continuing with the
experiment. Denature the tube for 10min at 70°C. Mix
the sample after 5 min.
- Pulse spin the tubes.
- Place tube 1 into a 37°C hot block and incubate
for 60min in the dark.
- Apply the contents of tube 2 onto the array grid
and rock the slide to ensure even coverage within the
well. Remove any air bubbles with a tip, but do not
touch the grid. Work quickly to minimise drying of the
- Transfer the slide into the humidity chamber
and place on the rocking table for 60min shaking at
- Pulse spin tube 1.
- Remove slide from humidity chamber.
- Apply DNA onto the slide. Remove any air bubbles.
- Rock the slide to mix the prehybridisation and
hybridisation solutions and to ensure even coverage
within the well.
- Place the slide into a slide mailer humidified with
- Seal well with Parafilm and place into the hybridisation
oven at 37°C.
- Incubate with gentle rocking (5rpm) for 48h,
turning the slide by 90° after 24h.
7. Scanning and Analysis
- Remove the slide from the incubator and increase
the temperature of the hybridisation oven up to
- Remove the rubber cement.
- Place the slide into a hellendahl jar containing
PBS/0.05% Tween 20 to wash off excess hybridisation
- Transfer the slide into a glass trough and wash in
PBS/0.05% Tween 20 for 10 min shaking at room
- Transfer the slide into a preheated trough containing
50% formamide/2x SSC at 42°C. Incubate at
42°C with shaking for 30 min.
- Transfer the slide into a trough of fresh PBS/0.05%
Tween 20 and incubate at room temperature with
- Transfer the slide into a metal rack and spin at
150g for 5min to dry.
- Store the slide in a light-proof box at room temperature
until ready to scan.
Our slides are scanned using an Axon 4000B
scanner (Axon Instruments, Burlingame, CA) and
the images are analysed by GenePix Pro 3.0 software
(Axon Instruments, Burlingame, CA). Spots are
defined using the automatic grid feature and adjusted
manually where necessary. Fluorescence intensities of
all spots are calculated after local background subtraction.
The ratios of the intensities are then plotted
in log2 scale against the distance of the clones along
the chromosomes from the p termini. Breakpoints are
identified by a shift in ratio intensities typically from
4 log 2 to-4 log 2 (or vice versa), with a spanning clone
showing an intermediate ratio close to 0 log 2.
Batches of Cot-1 DNA need to be tested before being
used routinely in experiments. At present we order
Cot-1 DNA from Roche (1581074) or Invitrogen (15279-
011) and perform test hybridisations on the array using
tumour cell line DNA with previously determined
characteristic genomic copy number changes.
During array hybridisation, care must be taken to
prevent the slides from drying out. Just before the
hybridisation solution is added to the slide, some of
the pre-hyb solution can be removed carefully with a
pipette tip. However, this increases the chances of the
slide drying out.
All gels used in this article are 1% agarose gels with
ethidium bromide. It is advised that these gels are run
before progression to the next step to check that each
process has been performed successfully.
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