Nucleic acid hybridization assays using cloned target DNA, and microarray hybridization technol

Section 4.2.5 colonies of bacteria or other suitable host cells which contain recombinant DNA can generally be selected or identified by the ability of the insert to inactivate a marker vector gene (e.g. b-galactosidase, or an antibiotic-resistance gene). A specific approach can be used when the desired recombinant DNA contains a DNA sequence that is closely related to an available nucleic acid probe. In the case of bacterial cells used to propagate plasmid recombinants, the cell colonies are allowed to grow on an agar surface and then transferred by surface contact to a nitrocellulose or nylon membrane, a process known as colony blotting (see Figure 5.18 ). Alternatively the cell mixture is spread out on a nitrocellulose or nylon membrane placed on top of a nutrient agar surface, and colonies are allowed to form directly on top of the membrane. In either approach, the membrane is then exposed to alkali to denature the DNA prior to hybridizing with a labeled nucleic acid probe.

After hybridization, the probe solution is removed, and the filter is washed extensively, dried and submitted to autoradiography using X-ray film. The position of strong radioactive signals is related back to a master plate containing the original pattern of colonies, in order to identify colonies containing DNA related to the probe. These can then be individually picked and amplified in culture prior to DNA extraction and purification of the recombinant DNA.

A similar process is possible when using phage vectors. The plaques which are formed following lysis of bacterial cells by phage will contain residual phage particles. A nitrocellulose or nylon membrane is placed on top of the agar plate in the same way as above, and when removed from the plate will constitute a faithful copy of the phage material in the plaques, a so called plaque-lift. Subsequent processing of the filter is identical to the scheme in Figure 5.18 .

Figure 5.19 ) and could be copied and distributed to numerous laboratories throughout the world.

Schena et al., 1998 ). Although reminiscent of the filter-based arrays, microarray construction involves quite different procedures. The surfaces involved are glass rather than porous membranes and the microarrays can be divided into two main classes according to their method of construction:

• Microarrays of DNA clones delivered by microspotting. Here the DNA clones have been prepared in advance and then printed onto the surface of a microscope slide (Figure 5.20A ). See Figure 20.6A for an application.
• Microarrays of oligonucleotides synthesized in situ. This approach has been pioneered by the company Affymetrix, Inc., in Santa Clara, CA, and typically involves a combination of photolithography technology from the semiconductor industry with the chemistry of oligonucleotide synthesis (Figure 5.20B ). See Figure 20.6B for an application.

As in the case of reverse dot-blotting ( Section 5.3.1 ), the DNA microarray technologies employ a reverse nucleic acid hybridization approach: the probes consist of unlabeled DNA fixed to a solid support (the arrays of DNA or oligonucleotides) and the target is labeled and in solution. Although the technology for establishing DNA microarrays was only developed in the last few years, already there have been numerous important applications and their impact on future biomedical research and diagnostic approaches is expected to be profound (see Box 5.5 ).

Figure 5.18. Colony hybridization involves replicating colonies on to a durable membrane prior to hybridization with a labeled nucleic acid probe. This method is popularly used to identify colonies containing recombinant DNA, should a suitable labeled probe be available.

Figure 5.20. Construction of DNA and oligonucleotide microarrays. (A ) Robotic spotting for construction of DNA microarrays. Left, a microarray robot, with a table configuration which contains 160 slides with four microtiter plates, two wash stations and the dryer. Right, a laser scanner showing the optical table, power supplies for the lasers and photomultiplier tube cooling, the Ludi stage and lenses (see Cheung et al., 1999 for more details). The microspotting of samples by robots can be performed by physical contact between spotting pins and the solid surface (of a microscope slide) or by an ink-jetting approach as is used in standard printing (the sample is loaded into a miniature nozzle equipped with a piezoelectric fitting and an electric current is used to expel a precise amount of liquid from the jet onto the substrate). Images kindly supplied by Aldo Massimi, Raju Kucherlapati and Geoffrey Childs at the Albert Einstein College of Medicine, New York. Reprinted from Cheung et al . (1999) Nature Genet ., 21 (suppl.), pp. 15 19, with permission from Nature America, Inc. (B ) Construction of an oligonucleotide microarray by combining photolithography and in situ synthesis of oligonucleotides. Oligonucleotides are synthesized in situ in sequential steps starting from a 3 mononucleotide which is anchored to the surface of a glass wafer. The photolithography entails modifying the glass wafer with photolabile protecting groups which can be eliminated when exposed to light and the use of carefully constructed photomasks which allow light to pass through onto carefully selected spatial coordinates. For those areas of the wafer which receive light passing through the photomask, the removal of the photolabile protective groups permits a new synthesis step. In this example thymidine is shown being coupled together with a protective photolabile group.