1. Growing and Making FISH Probes
Crista Illingworth
Sheffield Regional Cytogenetics Service Sheffield Children’s NHS Trust

2. Fluorescent in situ hybridisation (FISH)
FISH can be used to detect structural rearrangements,
gene amplifications, translocations, microdeletions.

3. 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

4. Many commercial fish probes are available for common disorders or abnormalities…
What happens for rare cases?

5. How to make your own probes
 Choosing DNA
 Growing and preparing DNA
 Labeling DNA
 QC

6. Case for which no commercial probe is available
Probe Preparation: From Start to Finish
Case for which no commercial probe is available

7. Identification of region of interest by G-banding, array or CGH
Case for which no commercial probe is available
Probe Preparation: From Start to Finish
Identification of region of interest by G-banding, array or CGH

8. Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Probe Preparation: From Start to Finish
Identify and order DNA
Identification of region of interest

9. How do you identify suitable DNA ?
The human genome browser Ensembl at the Sanger Centre

10. 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.
parB
parA
repE
oriS
CATr T7
Sp6
HindIII BamH1
Not1
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.

11. 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

12. Human genome project Chromosome
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 A
Contig B
Contig C
Contig D
Human genomic DNA cloned into BAC

13. Human genome project Clones assembled to produce a contig
Fragment size in a BAC library

14. DNA clones

15. Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
BAC arrives as E.coli
Identification of region of interest by G-banding

16. Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Probe Preparation: From Start to Finish
Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
Identify and order BAC
BAC arrives as E.coli
E.coli grown to amplify DNA
Identification of region of interest

17. The BAC vector contains Chloramphnicol 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
The BAC vector contains Chloramphnicol acetyl transferase
Cells are spun and collected

18. BAC DNA prepared from E.coli
Case for which no commercial probe is available
Probe Preparation: From Start to Finish
Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
Identify and order BAC
BAC arrives as E.coli
E.coli grown to amplify DNA
Identification of region of interest
BAC DNA prepared from E.coli

19. The principle of DNA preparation by Alkaline lysis from E.coli:
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

20. Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
BAC arrives as E.coli
E.coli grown to amplify DNA
Identification of region of interest
BAC DNA prepared from E.coli
BAC DNA prepared from E.coli
DNA fluorescently labelled

21. 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.

22. 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. 
DNase nicks DNA
DNA polymerase I removes individual bases from the 5’ end
DNA polymerase I adds new nucleotides to the 3’ hydroxyl

23. 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. 
DNase nicks DNA
DNA polymerase I removes individual bases from the 5’ end
DNA polymerase I adds new nucleotides to the 3’ hydroxyl
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.

24. DNA under goes quality control measures
Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
BAC arrives as E.coli
E.coli grown to amplify DNA
Identification of region of interest
BAC DNA prepared from E.coli
DNA under goes quality control measures
DNA fluorescently labelled

25. We have been using 3 methods: 1)PCR of STS markers
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.

26. 1) Sequence Tagged Sites Back to the human genome project…
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.

27. Possible that about 10% of BACs are incorrect.

28. 2) Finger print by restriction digest

29. 3) Hybridisation to metaphase chromosomes of control blood.
RP11-7H7 Chromosome 11
11 centromere

30. 745I14 extra signals
745I14 extra signals
1 q het
745I14

31. Probe Preparation: From Start to Finish
Case for which no commercial probe is available
Identify and order DNA
BAC arrives as E.coli
E.coli grown to amplify DNA
Identification of region of interest
BAC DNA prepared from E.coli
Case reported
DNA under goes quality control measures
Probe used to FISH case
DNA fluorescently labelled

33. Finding the Right BAC Suppliers of BACs
The human genome browser or Ensembl at the Sanger Centre allows you to search for BACs within the region of interest
Suppliers of BACs
Offer extensive coverage, cost of clone in addition to administration and delivery charge
Extensive coverage, cost of clone in addition to delivery charge
Clones are free, but sparse coverage