Growing and Making Growing and Making FISH ProbesFISH Probes

Crista IllingworthCrista Illingworth

Crista.Illingworth@SCH.NHS.UKCrista.Illingworth@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