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Growing and Making Growing and Making FISH ProbesFISH Probes
Crista IllingworthCrista Illingworth
[email protected]@SCH.NHS.UK
Sheffield Regional Cytogenetics Service Sheffield Children’s NHS
Trust
Fluorescent in situ hybridisation Fluorescent in situ hybridisation (FISH)(FISH)
FISH can be used to detect structural rearrangements, gene amplifications, translocations, microdeletions.
1) Cells are dropped on to a glass slide causing chromosomes to spread
2) Fluorescently labeled probe is placed on chromosomes and sealed.
3) The probe and chromosomes are denatured, hybridised then washed
4) Chromosomes are counter stained using DAPI
5) Slide is viewed under a fluorescent microscope
Many commercial fish probes Many commercial fish probes are available for common are available for common
disorders or abnormalities…disorders or abnormalities…
What happens for rare cases?
Choosing DNA
Growing and preparing DNA
Labeling DNA
QC
How to make your own probesHow to make your own probes
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
Case for which no commercial
probe is available
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish
Identification of region of
interest by G-banding, array
or CGH
Identify and order DNA Identification
of region of interest
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
How do you identify suitable DNA ?How do you identify suitable DNA ?The human genome browser
http://genome.uscs.edu/ Ensembl at the Sanger Centre http://www.ensembl.org/index
Bacterial Artificial ChromosomesBacterial 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.
parB
parA
repE
oriS
CATr
T7
Sp6
HindIII BamH1Not1
Not1
What is the DNA? What is the DNA? To make a suitable FISH probe we To make a suitable FISH probe we need a piece of DNA about 80 Kb.need a piece of DNA about 80 Kb.
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 NotIInserts can be transcribed using T7 or Sp6 promoters.
BACs are based on the BACs are based on the E.coliE.coli F-factor, the plasmid F-factor, the plasmid responsible for conjugation responsible for conjugation in in E.coliE.coli..
High stability, low rate of chimeric clones.
Low yield
Human genome projectHuman genome projectCloning 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
Contig A Contig B Contig C Contig D
Human genomic DNA cloned into BAC
Chromosome
Human genome projectHuman genome projectClones assembled to produce a contig
Fragment size in a BAC library
DNA clones
Identify and order DNAIdentification
of region of interest by G-
banding
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
BAC arrives as E.coli
Case for which no commercial
probe is available
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish
Identify and order BACIdentify and order DNAIdentification
of region of interest
BAC arrives as E.coli
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
E.coli grown to amplify DNA
The BAC vector contains The BAC vector contains Chloramphnicol acetyl transferaseChloramphnicol acetyl transferase
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
Cells are spun and collected
Case for which no commercial
probe is available
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish
Identify and order BACIdentify and order DNA
BAC DNA prepared from
E.coli
Identification of region of
interest
BAC arrives as E.coli
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
E.coli grown to amplify DNA
Preparation of BAC DNAPreparation of BAC DNAThe principle of DNA preparation by Alkaline lysis from E.coli:
1)Lysis
SDSSDS solubilizes phospholipids and proteins in cell membrane
NaOHNaOH denatures the chromosomal and BAC DNA
2) Neutralization
Acidic potassium acetateAcidic potassium acetate neutralizes the lysate .
High salt concentration causes potassium dodecyl sulphatepotassium 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 ethanolethanol (dehydrates surface of DNA) Put at -20oC
BAC DNA prepared from
E.coli
Identify and order DNA
DNA fluorescently
labelled
BAC DNA prepared from
E.coli
Identification of region of
interest
BAC arrives as E.coli
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
E.coli grown to amplify DNA
Nick translation labeling of FISH probesNick 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.
Nick translation labeling of FISH probesNick 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.
DNase nicks DNA
DNA polymerase I removes individual bases from the
5’ end
DNA polymerase I adds new nucleotides to the
3’ hydroxyl
Nick translation labeling of FISH probesNick 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.
Introduction of Nicks means DNA will break Introduction of Nicks means DNA will break into smaller and smaller pieces depending into smaller and smaller pieces depending on how long reaction is run for.on how long reaction is run for.
Optimum size of fragments is around
200 to 300 bp
DNase nicks DNA
DNA polymerase I removes individual bases from the
5’ end
DNA polymerase I adds new nucleotides to the
3’ hydroxyl
Identify and order DNA
DNA fluorescently
labelled
BAC DNA prepared from
E.coli
Identification of region of
interest
BAC arrives as E.coli
DNA under goes quality
control measures
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish Case for which no commercial
probe is available
E.coli grown to amplify DNA
How can we be sure we have the right BAC?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.
1) Sequence Tagged Sites1) 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.
Possible that about 10% of BACs are incorrect.
2) Finger print by restriction digest2) Finger print by restriction digest
3) Hybridisation to metaphase 3) Hybridisation to metaphase chromosomes of control blood.chromosomes of control blood.
RP11-7H7 Chromosome 11
11 centromere
745I14 extra signals
1 q het
745I14 extra signals
745I14
Identify and order DNA
DNA fluorescently
labelled
BAC DNA prepared from
E.coli
Identification of region of
interest
BAC arrives as E.coli
DNA under goes quality
control measures
Probe used to
FISH case
Probe Preparation: From Start to FinishProbe Preparation: From Start to Finish
Case reported
Case for which no commercial
probe is available
E.coli grown to amplify DNA
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.invitrogen.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