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Growing and Making FISH Probes Crista Illingworth Sheffield Regional Cytogenetics Service Sheffield Childrens NHS Trust.

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Presentation on theme: "Growing and Making FISH Probes Crista Illingworth Sheffield Regional Cytogenetics Service Sheffield Childrens NHS Trust."— Presentation transcript:

1 Growing and Making FISH Probes Crista Illingworth Sheffield Regional Cytogenetics Service Sheffield Childrens NHS Trust

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

3 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

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

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

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

7 Probe Preparation: From Start to Finish Identification of region of interest by G- banding, array or CGH

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

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

10 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 CAT r T7 Sp6 HindIII BamH1 Not1 What is the DNA? To make a suitable FISH probe we 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 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 Contig A Contig B Contig C Contig D Human genomic DNA cloned into BAC Chromosome

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

14 DNA clones

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

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

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 37 o C overnight. Single colony selected and grown in 10 ml LB with chloramphenicol at 37 o C overnight with shaking Cells are spun and collected

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

19 Preparation of BAC DNA The principle of DNA preparation by Alkaline lysis from E.coli: 1)Lysis SDS SDS solubilizes phospholipids and proteins in cell membrane NaOH NaOH denatures the chromosomal and BAC DNA 2) Neutralization Acidic potassium acetate Acidic potassium acetate neutralizes the lysate. potassium dodecyl sulphate 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 ethanol Using high salt –vely charged DNA is able to clump when +ve salt ions are added) and ethanol (dehydrates surface of DNA) Put at -20 o C

20 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 Finish Case for which no commercial probe is available E.coli grown to amplify DNA

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. Introduction of Nicks means DNA will break into smaller and smaller pieces depending 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

24 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 Finish Case for which no commercial probe is available E.coli grown to amplify DNA

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 centromere

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

31 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 Finish Case reported Case for which no commercial probe is available E.coli grown to amplify DNA

32

33 Finding the Right BAC 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


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