Automation of array-CGH · commonly named array-CGH (4, 5). Array-CGH principle In array-CGH, DNA...
Embed Size (px)
Transcript of Automation of array-CGH · commonly named array-CGH (4, 5). Array-CGH principle In array-CGH, DNA...
Automation of array-CGH
Tecan HS™ Hybridization Stations
Introduction Comparative genomic hybridization (CGH) was introduced in 1992 for genome wide analysis of DNA copy number changes (1). The main appli-cation of this technology has been in the study of solid tumors where it is extremely difficult to produce metaphase chromosomes for direct analysis of rearrangements (2). However, the resolution with which copy number changes can be detected is limited (to 3-5 Mb) by the use of normal metaphase chromosomes as hybridization substrates (3). As a result of the public effort to sequence the human genome, it became possible to replace the metaphase chromosomes with an arrayed series of well mapped (and sequenced) large-insert clones spotted onto glass slides. This methodology, first described in the late nineties, is now commonly named array-CGH (4, 5).
Array-CGH principle In array-CGH, DNA from a test genome is fluo-rescently labeled in one color (e.g. green) and co-hybridized with a reference genome labeled
in a second color (e.g. red) onto clone DNA spotted on glass slides (Figure 1). The labeled probes are hybridized in the presence of Cot1 DNA to suppress repetitive elements and allow the hybridization to represent mainly unique sequences in the genome. Where there is equal copy number between the two genomes, equal amounts of test and reference DNA hybridize and the corresponding spotted DNA appears as a mixture of both colors (i.e. yellow when mixture of green and red). Where a region of the test genome is deleted, the corresponding spotted DNA is hybridized with more reference DNA than test DNA and thus appears red. Where a region of the test genome is gained, the corresponding spotted DNA hybridizes more test DNA than reference DNA and appears green. By measuring the ratio of the fluorescence intensities for each spot (mapped cloned DNA), ratio profiles are generated where a high ratio indicates copy number gain and a low ratio copy number loss (Figure 1).
Figure 1. Principle of array-CGH (Comparative genomic hybridization) The initial microarray constructed at The Well-come Trust Sanger Institute covers the entire genome with an average of one clone every megabase (6). It has been shown that this array can quantify both small genome copy number
changes of less than a single copy and amplifications of at least 60 fold. One example of CGH profile generated with this array is de-scribed in Figure 2.
Figure 2. Array-CGH analysis of the 769P cancer cell line with one large-insert clone microarray covering the entire genome at 1 Mb resolution. (A) Composite image of the microarray after co-hybridization of the 769P cell line DNA probe (green -labeled in Cy3) with a normal reference DNA (red - labeled in Cy5). This microarray is composed of 3523 clones, each spotted in duplicate. (B) Chromosome 1 profile showing a single copy number loss of the distal part of the short arm of chromosome 1 (dots in red) and a single copy gain of the distal part of the long arm (dots in green). (C) Whole genome profile showing additional losses on chromosome 3p, 9p, 1q and 14 (red) and gains on chromosome arms 5q and 8q (green).
The Wellcome Trust Sanger Institute has re-cently published the screening of micro-dele-tions/duplications in 50 patients with learning disability and dysmorphic features using this microarray (7). In this initial study, array-CGH detected subtle copy number changes - not visible by conventional cytogenetics - in 12 patients (24%). These results demonstrated the power of array CGH for systematic screening of constitutional chromosomal copy number changes. As a consequence, increasing num-bers of clinical laboratories are now considering the implementation of array-CGH as diagnostic tool for patients with dysmorphology.
The success of this initial study led to the plan-ning of new research projects involving larger cohorts of patients. The first screening was carried out exclusively by manual hybridizations and subsequent washes. While these original protocols gave satisfactory results, they re-quired a high level of expertise and were time-consuming. Thus protocols have been adapted for using the Tecan HS 4800™ hybridization station and a new procedure was designed enabling automated hybridization and washes of large insert clone microarrays in less than 24 hours on this machine. The standard protocol resulting from this work is described below.
Methods Labeling DNA is labelled using the Bioprime labeling kit (Invitrogen Co.) with modifications of the nucleotide mix. Briefly, a 130.5 µl reaction is set up containing 150 ng of DNA and 60 µl of 2.5x random primer solution. After denaturing the DNA for 10 minutes at 100°C, 15 µl of 10x dNTP mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, and 2 mM dTTP in TE buffer), 1.5 µl of 1 mM Cy5-dCTP or Cy3-dCTP (NEN Life Science Products), and 3 µl of Klenow fragment are added on ice to a final reaction volume of 150ml. The reaction is incubated at 37°C overnight and stopped by adding 15 µl of stop buffer supplied in the kit. Unincorporated nucleotides are removed by use of Microcon YM-300 Filter Devices (Millipore Co.), according to the suppliers’ instructions.
Sample preparation and hybridization Cy3 and Cy5 labeled test and control DNAs are combined, precipitated together with 135 µg of human Cot1 DNA (Roche) and resuspended in 120 µl of hybridization buffer (50% formamide, 5% dextran sulphate, 0.1% Tween 20, 2xSSC and 10 mM Tris-HCl, pH 7.4). Pre-hybridization solution is prepared simultaneously by precipitating 80 µl of herring sperm DNA (10 mg/ml, Sigma Aldrich) and re-suspending it in 120 µl of hybridization buffer. The pre-hybridization and hybridization solution are then denatured for 10 min. at 72°C. The pre-hybridization solution is then injected into the Tecan chamber following instructions displayed on the station. During pre-hybridization, the hybridization solution is incubated at 37°C until its injection, still following displayed instruction. The program and liquids used on the HS 4800 hybridization station are described on Figure 3.
Figure 3. Current standard protocol for CGH on large-insert clone microarrays on the HS 4800 hybridization station. On this program, the washing solutions are: [ch.1] PBS with 0.05% Tween 20; [ch.3] 0.1x SSC; [ch.5] HPLC-purified water.
This procedure gives very reliable results, comparable in quality to those obtained by manual procedure (Figure 4). In conse-quence, progressively most of the hybridi-zations made at The Wellcome Trust Sanger
Institute have been transferred onto multiple Tecan HS 4800 hybridization stations, which enable the processing of up to 60 slides per week and per unit.
Figure 4. Copy number measurement by array-CGH on the Tecan HS 4800 hybridization station, compared to alternative manual procedures. Array-CGH was performed on the 769P cell line using the 1-Mb whole genome microarray on the HS 4800 hybridization station (orange dots) compared to alternative manual procedures (dark and light blue dots). Data points for chromosome 1 were extracted and sorted by known DNA copy numbers: 1 (n=28), 2 (n=118) or 3 (n=90). The automated procedure gave log2 ratio values closer to the expected values of -1 and 0.58 in the presence of 1 and 3 copies versus 2 in the normal reference DNA, respectively. In addition, no increased variability was observed between data points located in the normal genomic segment (2 copies versus 2, log2 ratio value of 0). These results illustrate the effectiveness of array-CGH automation.
Perspectives A new version of the whole-genome microarray has recently been developed at The Wellcome Trust Sanger Institute. This array, composed of 27,000 large-insert clones, covers the entire human genome at tiling path resolution and enables the screening of copy number changes at a resolution down to less than 100 kilobases. The Wellcome Trust Sanger Institute has initi-ated a comprehensive survey of copy number variation in the human population by high throughput CGH analysis of DNA samples from
anonymous individuals on this array. For the first phase of this project, more than 500 hybridizations have been processed on the HS 4800 hybridization station in six months, with an estimated success rate of more than 80%. One typical comparison between two anonymous human genomes with the tiling path array en-ables the detection of more than 100 deletions or duplications in one individual DNA compared to the other (Figure 5).
Figure 5. Genome wide comparison of 2 anonymous normal DNA samples by array-CGH with the Sanger whole genome tiling path array. (A) Composite image of the microarray after co-hybridization of two normal genomic DNA probes (one labeled in Cy3, green the other in Cy5, red). This array is composed of 27,000 clones, each spotted once. (B) Whole genome profile generated after fusion of the results from two independent hybridizations performed in dye-swap. DNA copy number changes are showed in green (gain in test DNA or loss in reference DNA) and red (loss in test DNA or gain in reference DNA). (C) CGH profiles for chromosomes 8 and 18. Please note the large duplication on the 8p arm (in green, log2 values around 0.5) and the small deletion on 18q (in red, log2 values down to -0.75). This result illustrates the high level of DNA copy number variation within the human population. This type of variation between normal individu-als has been underestimated in the past, mainly because of the low resolution of previously existing screening methods compared to DNA microarray technology. The high throughput application of array-CGH for whole genome screening of copy number variation will help understanding its role in human evolution and disease. This would not have been possible without the full standardization of this method, which required its automation.
Conclusion Compared with conventional comparative ge-nomic hybridization (CGH), the array-based approach (array-CGH) provides an increased
potential for higher resolution and wider dynamic range results. The array format offers better possibilities for automation, which is a key factor for standardization, improving reproduci-bility and increasing throughput. Automation reduces the level of expertise re-quired to run array-CGH experiments and also helps to reduce variability in the critical assay steps, like washing and drying. The results presented in this study show that the automated procedure gives values closer to the expected values compared to the manual procedure. It is possible that this derives from higher stringency in the hybridization process through effective liquid mixing and more efficient washing in the automated procedure. In summary these results clearly illustrate the effectiveness and benefits of array-CGH auto-mation.
References 1. Kallioniemi, A., Kallioniemi, O.P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F., and Pinkel, D. (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818-21. 2. Forozan, F., Karhu, R., Kononen, J., Kallioniemi, A., and Kallioniemi, O.P. (1997) Genome screening by comparative genomic hybridization. Trends Genet. 13:405-9. 3. Lichter, P., Joos, S., Bentz, M., and Lampel, S. (2000) Comparative genomic hybridization: uses and limitations. Semin Hematol 37:348-57. 4. Solinas-Toldo, S., Lampel, S., Stilgenbauer, S., Nickolenko, J., Benner, A., Dohner, H., Cremer, T., and Lichter, P. (1997) Matrix-based comparative genomic hybridiza-tion: biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20:399-407. 5. Pinkel, D., Segraves, R., Sudar, D., Clark, S., Poole, I., Kowbel, D., Collins, C., Kuo, W.L., Chen, C., Zhai, Y., Dairkee, S.H., Ljung, B.M., Gray, J.W., and Albertson, D.G. (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridiza-tion to microarrays. Nat Genet 20:207-11.
6. Fiegler, H., Carr, P., Douglas, E.J., Burford, D.C., Hunt, S., Scott, C.E., Smith, J., Vetrie, D., Gorman, P., Tomlinson, I.P., and Carter, N.P. (2003) DNA microarrays for com-parative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36:361-74. 7. Shaw-Smith, C., Redon, R., Rickman, L., Rio, M., Willatt, L., Fiegler, H., Firth, H., Sanlaville, D., Winter, R., Colleaux, L., Bobrow, M., and Carter, N.P. (2004) Microarray based comparative genomic hybridization (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 41:241-8.
Acknowledgement Tecan would like to thank the following persons for performing the experiments and providing their data: Richard Redon, Nigel Carter at the Wellcome Trust Sanger Institute, Cambridge, UK (http://www.sanger.ac.uk/). For further information regarding the data in this publication please contact Dr. Richard Redon at The Wellcome Trust Sanger Institute (mailto:[email protected]).
Note: The applications described here are not approved in the US for clinical diagnostic purposes. Tecan Group Ltd. is making all efforts to include accurate and up-to-date information into this publication. Yet, it cannot be ruled out that omissions or errors might have occurred. Therefore, Tecan Group Ltd. cannot make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. © 2005, Tecan Trading AG, Switzerland, all rights reserved. Tecan® is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. HS™, HS 4800™ are trademarks of Tecan Group Ltd., Männedorf, Switzerland Tecan Asia (Pte) Ltd., T +65 644 41 886, Tecan Sales Austria GmbH, T +43 62 46 89 33, Tecan Sales International GmbH, T +43 62 46 89 33, Tecan Benelux B.V.B.A., T +32 15 42 13 19, Tecan Benelux B.V.B.A., T +31 18 34 48 17 4, Tecan Group Ltd., Beijing Rep. Office, T +86 10 586 95 936, Tecan Deutschland GmbH, T +49 79 51 94 170, Tecan France S.A.S., T +33 4 72 76 04 80, Tecan Italia S.r.l., T +39 02 215 21 28, Tecan Sales International GmbH, T +43 62 46 89 33, Tecan Japan Co. Ltd, T +81 44 556 7311, Tecan Nordic AB, T +46 31 75 44 000, Tecan Nordic AB, Rep. Office Denmark, T +45 70 234 450, Tecan Portugal, T +351 21 000 82 16, Tecan Sales Switzerland AG, T +41 44 922 89 22, Tecan Iberica Instr. S.L., T +34 93 490 01 74, Tecan UK Ltd., T +44 11 89 300 300, Tecan US Inc., T +1 919 361 5200 www.tecan.com 6