Growing and Making FISH Probes

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

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

7. 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

8. 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 http://genome.uscs.edu/ Ensembl at the Sanger Centre http://www.ensembl.org/index

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

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 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 B Contig A 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 BAC arrives as E.coli Identify and order DNA Identification of region of interest by G-banding

16. Case for which no commercial probe is available Identification of region of interest Identify and order BAC Identify and order DNA BAC arrives as E.coli 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

17. 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. Case for which no commercial probe is available Identification of region of interest Identify and order BAC Identify and order DNA BAC arrives as E.coli 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

19. Preparation of BAC DNA The principle of DNA preparation by Alkaline lysis from E.coli: 1)Lysis SDSsolubilizes 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 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

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

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

24. 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

25. 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 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 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

33. 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