Human Genome Project Resources for Breakpoint Mapping
The announcement in April 2003 that the Human Genome Project was complete and was available publicly through intuitive genome browsers (e.g., see http://www.ensembl.org/Homo_sapiens/ and http://genome.ucsc.edu/cgi-bin/hgGateway) revolutionised experimental genome analysis. More precisely the study of chromosome rearrangements has been facilitated by the availability of easily accessible, ordered, fully sequenced tiling path clones covering the entire human genome. This resource enables chromosome breakpoints to be mapped more rapidly and with increased confidence.
Conventionally a chromosome rearrangement has been mapped by walking along a chromosome sequentially hybridising fluorescently labelled DNA clones (FISH) until the signal is no longer retained on one derivative chromosome but is seen on the reciprocal derivative chromosome. The genomic position of the breakpoint is then delineated by these two clones and the spanning clone can be found by FISHing clones at a higher resolution (McMullan et al., 2002). The genome annotation also now easily available on web browsers (e.g., http://vega.sanger.ac.uk/ Homo_sapiens/) provides an additional level of information regarding the gene content of a genomic region of interest. Both known and predicted genes are detailed along with additional information, including repeat content, GC content, mouse homology, and many additional features. This information, together with the identification of breakpoint spanning clones linked directly to these databases, allows candidate genes involved in the phenotype of a patient to be identified readily.
Clones sequenced as part of the Human Genome Project are available readily to international researchers for minimal cost (e.g., BACPAC resources: http://bacpac.chori.org/, Invitrogen: http://clones. invitrogen.com/, and the Resources for Molecular Cytogenetics, University of Bari: http://www.biologia. uniba.it/rmc/). The sequence obtained from these clones is freely accessible (e.g., http://www.ncbi.nlm. nih.gov/genome/clone/, http://www.ensembl.org/ Homo_sapiens/, and http://genome.ucsc.edu/cgi-bin/hgGateway).
The availability of such a comprehensive set of clones has not only greatly facilitated conventional approaches to breakpoint mapping studies but has also enabled the development of genome-wide, large insert clone DNA microarrays. The selection, DNA extraction, and labelling of clones for multiple sequential DNA clone in situ hybridisation experiments required to map breakpoints are often time-consuming processes. However, the clones can be used as ordered targets themselves for breakpoint mapping after gridded assembly on a DNA microarray (Fiegler et al., 2003a). DNA microarrays have been recently developed mostly with the aim of studying genome imbalance in tumour DNA samples (Solinas-Toldo et al., 1997). We have exploited this resource to study structural chromosome rearrangements, including rearrangements with no genomic imbalance. This type of analysis, termed array painting (Fiegler et al., 2003b), involves isolating derivative chromosomes away from other chromosome homologues by flow sorting (Carter, 1994) or microdissection prior to hybridisation onto the DNA microarrays.
The limit of array painting as a tool for mapping chromosome breakpoints is defined by the resolution of the clones gridded. At present we use a 1-Mb array to define the breakpoints to within a megabase (see Fig. 3) and then use conventional FISH methods (see article by Leversha) to refine the breakpoints and find the spanning clones. Clones that span the breakpoint show intermediate ratios on the array (see Fig. 2). Statistically, 1 in every 10 breakpoints will have spanning clones identified directly on the 1-Mb resolution array.
This technique has proven to be invaluable in the detailed study of chromosome rearrangements. When used in conjunction with array CGH, it can give an insight into rearrangements that might have been undetected by G-banding analysis. Increased array resolution, e.g., a tiling path array, in the study of simple chromosome rearrangements would identify spanning clones within a single experiment.
II. MATERIALS AND INSTRUMENTATION
A. Purification of Flow-Sorted DNA
N-Lauroylsarcosine sodium salt (Sigma, L5125)
Proteinase K (Invitrogen, 25530-015)
Phenylmethylsulfonyl fluoride (PMSF) (Sigma, P7626)
Pellet Paint nonfluorescent coprecipitant (Novagen, 70748)
B. Random Labeling of DNA for Array Painting
BioPrime DNA labeling kit (Invitrogen, 18094-011) contains 2.5× random primers solution, sterile water, Klenow fragment, and stop buffer
dNTPs (Amersham Biosciences, 27-2035-02)
Cyanine 3-dCTP (Perkin Elmer, NEL576001EA)
Cyanine 5-dCTP (Perkin Elmer, NEL577001EA)
Microspin G50 columns (Amersham Biosciences, 27- 5330-01)
C. Array Hybridization
Herring sperm DNA (Sigma, D7290)
Human Cot-1 DNA (Roche, 1581074)
Deionised formamide for hybridisation buffer (Sigma, F9037)
Dextran sulphate sodium salt (Amersham Biosciences, 17-0340-02)
HPLC water (VWR International, 152736D)
Rubber cement (Weldtite, 02002)
Formamide (Sigma, 47670)
Yeast tRNA (Invitrogen, 15401-029)
Tween 20 (VWR International, 663684B)
Hellendahl jars (RA Lamb, E95)
Glass troughs (RA Lamb, E106 + E98)
Parafilm (Sigma, P7793)
Shake "n" stack hybridisation oven (Thermo Hybaid, HBMOJCT)
A. Purification of Flow-Sorted DNA
Handle tubes containing flow-sorted material very gently!
Flow-sorted chromosomes come in batches of approximately 250,000 per 1.5ml microfuge tube, sorted into chromosome sheath buffer [10mM Tris-HCl (pH 8.0), 1mM EDTA, 100mM NaCl, 0.5 mM Na azide]. Sort 250,000 chromosomes into a volume of approximately 300µl. Amounts of reagents will vary according to the quantity of sorted sample.
B. Random Labeling of DNA for Array Palnting Solutions
1. Labeling Reactions
2. Clean-up of Labeling Reactions
Use 3 columns per labelling reaction. Once the columns have been prepared, they must be used immediately to avoid the resin drying out.
Sample Application Steps
C. Array Hybridization
2. Preparation of Slide
3. Preparation of Humidity Chambers
7. Scanning and Analysis
Our slides are scanned using an Axon 4000B scanner (Axon Instruments, Burlingame, CA) and the images are analysed by GenePix Pro 3.0 software (Axon Instruments, Burlingame, CA). Spots are defined using the automatic grid feature and adjusted manually where necessary. Fluorescence intensities of all spots are calculated after local background subtraction. The ratios of the intensities are then plotted in log2 scale against the distance of the clones along the chromosomes from the p termini. Breakpoints are identified by a shift in ratio intensities typically from 4 log 2 to-4 log 2 (or vice versa), with a spanning clone showing an intermediate ratio close to 0 log 2.
Batches of Cot-1 DNA need to be tested before being used routinely in experiments. At present we order Cot-1 DNA from Roche (1581074) or Invitrogen (15279- 011) and perform test hybridisations on the array using tumour cell line DNA with previously determined characteristic genomic copy number changes.
During array hybridisation, care must be taken to prevent the slides from drying out. Just before the hybridisation solution is added to the slide, some of the pre-hyb solution can be removed carefully with a pipette tip. However, this increases the chances of the slide drying out.
All gels used in this article are 1% agarose gels with ethidium bromide. It is advised that these gels are run before progression to the next step to check that each process has been performed successfully.
Carter, N. R (1994). Bivariaee chromosome analysis using a commercial flow cytometer. Methods Mol. Biol. 29, 187-204.
Fiegler, H., Carr, R, Douglas, E. J., Burford, D. C., Hunt, S., Smith, J., Vetrie, D., Gorman, R, Tomlinson, I. R M., and Carter, N. R (2003a). DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36, 361-374.
Fiegler, H., Gribble, S. M., Burford, D. C., Carr, R, Prigmore, E., Porter, K. M., Clegg, S., Crolla, J. A., Dennis, N. R., Jacobs, R, and Carter, N. P. (2003b). Array painting: A method used for the rapid analysis of aberrant chromosomes using DNA microarrays. J. Med. Genet. 40, 664-670.
McMullan, T. E W., Crolla, J. A., Gregory, S. G., Carter, N. R, Cooper, R. A., Howell, G. R., and Robinson, D. O. (2002). A candidate gene for congenital bilateral isolated ptosis identified by molecular analysis of a de novo balanced eranslocation. Hum. Genet. 110, 244-250.
Solinas-Toldo, S., Lampei, S., Stilgenbauer, S., Nickolenko, J., Benner, A., Döhner, H., Cremer, T., and Licheer, P. (1997). Matrix-based comparative genomic hybridization: Biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20, 399-407.
Telenius, H., Carter, N. P., Bebb, C. E., Nordenskj61d, M., Ponder, B. A. J., and Tunnacliffe, A. (1992). Degenerate oligonucleoeideprimed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13, 718-725.
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