Growing and Making FISH Probes

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Growing and Making FISH Probes
Crista Illingworth
Sheffield Regional Cytogenetics Service
Sheffield Childrens NHS Trust
Crista.Illingworth_at_SCH.NHS.UK
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Fluorescent in situ hybridisation (FISH) FISH
can be used to detect structural rearrangements,
gene amplifications, translocations,
microdeletions.
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3) The probe and chromosomes are denatured,
hybridised then washed
1) Cells are dropped on to a glass slide causing
chromosomes to spread
2) Fluorescently labeled probe is placed on
chromosomes and sealed.
4) Chromosomes are counter stained using DAPI
5) Slide is viewed under a fluorescent microscope
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Many commercial fish probes are available for
common disorders or abnormalities
What happens for rare cases?
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How to make your own probes
? Choosing DNA ? Growing and preparing DNA ?
Labeling DNA ? QC
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
Identification of region of interest by
G-banding, array or CGH
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Probe Preparation From Start to Finish
Identify and order DNA
Identification of region of interest
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How do you identify suitable DNA ?
The human genome browser http//genome.uscs.edu/
Ensembl at the Sanger
Centre http//www.ensembl.org/index
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What is the DNA? To make a suitable FISH probe
we need a piece of DNA about 80 Kb.
Bacterial Artificial Chromosomes Owing to the
low BAC copy number, the insert length that can
be recovered in BAC clones is usually much larger
than for other cloning systems. BAC clones thus
can be used for construction of libraries
covering genomes with a relatively small number
of stable E. coli clones.
oriS and repE elements mediate replication.
parA and parB maintain copy number at one or two
per genome. CATr provides a means of selection.
Insert DNA is cloned into the BamHI and HindIII
sites and excised using NotI Inserts can be
transcribed using T7 or Sp6 promoters.
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BACs are based on the E.coli F-factor, the
plasmid responsible for conjugation in E.coli.
High stability, low rate of chimeric clones.
Low yield
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Human genome project
Cloning a whole genome begins by amassing a
library of randomly cloned inserts. A set of
overlapping clones is called a contig. Contigs
represent cloned "islands" of the genome. As
more clones are characterized, contigs enlarge
and merge into one another.
Chromosome
Chromosome
Contig C
Contig D
Contig A
Contig B
Human genomic DNA cloned into BAC
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Human genome project
Clones assembled to produce a contig
Fragment size in a BAC library
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DNA clones
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
BAC arrives as E.coli
Identify and order DNA
Identification of region of interest by G-banding
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Probe Preparation From Start to Finish
Probe Preparation From Start to Finish
Case for which no commercial probe is available
E.coli grown to amplify DNA
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The E.coli arrive as a stab which is spread on LB
agar with chloramphenicol and grown at 37oC
overnight.
Single colony selected and grown in 10 ml LB with
chloramphenicol at 37oC overnight with shaking
The BAC vector contains Chloramphnicol acetyl
transferase
Cells are spun and collected
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Probe Preparation From Start to Finish
Probe Preparation From Start to Finish
Case for which no commercial probe is available
E.coli grown to amplify DNA
BAC DNA prepared from E.coli
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Preparation of BAC DNA
The principle of DNA preparation by Alkaline
lysis from E.coli 1)Lysis SDS solubilizes
phospholipids and proteins in cell membrane NaOH
denatures the chromosomal and BAC DNA 2)
Neutralization Acidic potassium acetate
neutralizes the lysate . High salt concentration
causes potassium dodecyl sulphate to precipitate
along with denaturated proteins, chromosomal DNA
and cell debris. Circular DNA is covalently
closed and is able to renature correctly so
staying in solution. 3) Clearing Precipitated
debris is cleared by centrifugation 4)
Precipitation Using high salt vely charged DNA
is able to clump when ve salt ions are added)
and ethanol (dehydrates surface of DNA) Put at
-20oC
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
BAC arrives as E.coli
E.coli grown to amplify DNA
Identify and order DNA
Identification of region of interest
BAC DNA prepared from E.coli
BAC DNA prepared from E.coli
DNA fluorescently labelled
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Nick translation labeling of FISH probes
mammalian dexyribonuclease (DNase I) hydrolyzes
double stranded DNA leaving random gaps with free
3 hydroxyl groups. E.coli DNA polymerase I.
removes individual bases from the 5 end, adds
new nucleotides to the 3 hydroxyl and 3 to 5
proof reading activity.
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Nick translation labeling of FISH probes
mammalian dexyribonuclease (DNase I) hydrolyzes
double stranded DNA leaving random gaps with free
3 hydroxyl groups. E.coli DNA polymerase I.
removes individual bases from the 5 end, adds
new nucleotides to the 3 hydroxyl and 3 to 5
proof reading activity.
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Nick translation labeling of FISH probes
mammalian dexyribonuclease (DNase I) hydrolyzes
double stranded DNA leaving random gaps with free
3 hydroxyl groups. E.coli DNA polymerase I.
removes individual bases from the 5 end, adds
new nucleotides to the 3 hydroxyl and 3 to 5
proof reading activity.
Optimum size of fragments is around 200 to 300 bp
Introduction of Nicks means DNA will break into
smaller and smaller pieces depending on how long
reaction is run for.
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
BAC arrives as E.coli
E.coli grown to amplify DNA
Identify and order DNA
Identification of region of interest
BAC DNA prepared from E.coli
DNA under goes quality control measures
DNA fluorescently labelled
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How can we be sure we have the right BAC?
We have been using 3 methods 1)PCR of STS
markers 2) Finger print by restriction digest 3)
Hybridisation to metaphase chromosomes of control
blood.
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1) Sequence Tagged Sites
What are STS? Back to the human genome project
Sequence Tagged Site. An STS is a short DNA
segment which is present at only one location in
the genome and whose sequence is known. Knowing
the sequence makes it possible to design a PCR
reaction to test for its presence in any sample.
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Possible that about 10 of BACs are incorrect.
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2) Finger print by restriction digest
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3) Hybridisation to metaphase chromosomes of
control blood.
RP11-7H7 Chromosome 11 11 centromere
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745I14 extra signals
745I14 extra signals
1 q het
745I14
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Probe Preparation From Start to Finish
Case for which no commercial probe is available
BAC arrives as E.coli
E.coli grown to amplify DNA
Identify and order DNA
Identification of region of interest
BAC DNA prepared from E.coli
Case reported
DNA under goes quality control measures
DNA fluorescently labelled
Probe used to FISH case
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(No Transcript)
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Finding the Right BAC
The human genome browser http//genome.uscs.edu/
or Ensembl at the Sanger
Centre http//www.ensembl.org/index allows you
to search for BACs within the region of interest
Suppliers of BACs
http//bacpac.chori.org/

Offer
extensive coverage, cost of clone in addition to
administration and delivery charge www.clones.invi
trogen.com/cloneranger

Extensive coverage, cost of
clone in addition to delivery charge
www.sanger.ac.uk/teams/teams63/clonerequest

Clones are free, but
sparse coverage