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The spatial resolution of microarray-based comparative genomic hybridization (array-CGH) is dependent on the length and density of target DNA sequences covering the chromosomal region of interest. Here we describe the methods developed at the Wellcome Trust Sanger Institute (Cambridge, UK) to construct microarrays composed of large-insert clones available through genome sequencing projects. These methods are applicable to Bacterial and Phage Artificial Chromosomes (BAC and PAC) as well as fosmid and cosmid clones. The protocols are scalable for the construction of microarrays composed of several hundreds up to several ten thousands clones.
Constructing large-insert clone arrays originally involved DNA extraction of DNA inserts from large volumes of bacterial cultures, resulting in only small quantities of DNA available for spotting (1-3). Although this strategy is practical for the construction of small microarrays composed of tens to hundreds of clones, it is not scalable to arrays composed of thousands of clones, such as arrays covering the whole genome with one clone every megabase or with tiling path coverage.
In order to amplify with high fidelity small amounts of large-insert clone DNA obtained from high throughput extraction of small bacterial cultures, several strategies have been developed, such as strand-displacement rolling circle amplification (4) or ligation-mediated PCR amplification (5). Here we describe the protocols for the use of degenerate oligonucleotide-primed PCR (DOP-PCR) (6).
DOP-PCR was originally designed for species-independent general DNA amplification (7). Used with a cycling protocol incorporating a small number of initial low temperature annealing cycles, DOP-PCR allows a general amplification of any target DNA i.e. a chromosomal segment, whole chromosome or whole genome. The target DNA is amplified by PCR with a mix of primers composed of unique 5′ and 3′ end sequences flanking a random variable hexanucleotide sequence (7).
Although DOP-PCR was designed for general amplification of any DNA fragment, the representation of the target DNA by the DOP-PCR products relies on the six fixed nucleotides located at the 3′ end of the primer sequence. In our protocols, each clone is amplified with three different sets of degenerate primers to improve the representation of large-insert clone sequence after DOP-PCR amplification. Each degenerate primer is designed with a different fixed 3′ hexanucleotide sequence, which has been selected for its presence at high frequency in the human genome (6).
In addition, because DNA preparations of human large-insert clones from bacterial cultures are contaminated with Escherichia coli genomic DNA (8), the three primer sets have been selected to be inefficient in the amplification of Escherichia coli DNA. In consequence, the PCR products using these three primer set contain mostly human insert sequences, which will greatly increase the quantity of printed DNA able to hybridize with the labeled test and reference genomic probes (6).
The three degenerate primers contain the same 5′ decanucleotide sequence. This property enables a secondary PCR amplification with one universal 5′-modified primer whose 3′ end matches the 5′ end of all DOP primers. The secondary PCR presents two advantages: (i) it increases exponentially the quantity of large-insert clone DNA available for array printing; (ii) it facilitates the incorporation of reactive groups for covalent attachment of the amplified DNA to specially coated glass slides. In our protocol, we use 5′ amino-linked secondary primers such that amplified large-insert clone DNA will bind covalently to CodeLink® activated slides (6).
The protocol in this section was developed at the Microarray Facility of the Sanger Institute (http://www.sanger.ac.uk/Projects/Microarrays/arraylab/methods.shtml) where it is routinely used to array DOP-PCR amplified DNA onto CodeLink™ activated slides (GE Healthcare), using Microgrid II stations (Biorobotics). Alternative strategies of microarray printing are described in more details in Chapters 5-7.
The authors would like to thank Heike Fiegler as well as Cordelia Langford and David Vetrie for their strong contributions in establishing the methods described in this chapter. This work was supported by the Wellcome Trust.
1Because DOP-PCR is a very efficient method for the amplification of template DNA from any organism, it is very sensitive to any trace of contaminating DNA. Always ensure that you are working in a sterile and DNA-free environment, i.e. in a PCR hood. Always work with materials and reagents (water, TAPS2 buffer and 1% W1) that have been UV-treated prior to use; do not UV-treat the AmpliTaq polymerase, dNTPs and primers.
2After DNA extraction, each well should contain approximately 200ng of DNA dissolved in 5μl of T0.1E. Add 195μl of water in each well before setting up PCR reactions.
3Negative controls should be included in every PCR set up. For each DOP primer template DNA should be replaced by equivalent volume of water at several positions in 96-well plates to ensure that there is no unexpected DNA contamination. For aminolink-PCR, if combined products from primary DOP-PCR negative controls are used as templates in negative controls, some DNA bands will be detected at corresponding positions after gel electrophoresis (Fig. 1B). However, these DNA fragments are smaller in size than those detected after using DOP-PCR products as templates: their presence is due to the formation of primer oligomers during the first PCR in negative controls, which are then amplified and extended during the secondary PCR reaction. Also include negative controls with only water as template in every secondary PCR set-up and check that there is no DNA product for these reactions.
4The 4x spotting buffer is specifically designed to print arrays on CodeLink® activated slides with Microgrid II stations. To print array on slides with another surface chemistry and/or using another printing station, refer to the documentation from the manufacturers.
5Long incubations with high humidity levels are used to facilitate covalent binding between the activated surface of the slide and the 5′ amino groups of printed DNA elements.
6Slides can be stored in the dark under dehumidified atmosphere for at least 3 months without loss of performance in hybridizations. We recommend the use of a sealed desiccation cabinet.