[41 Functional Genomics High Density Oligonucleotide Arrays

By Sashwati Roy, Savita Khanna, Kimberly Bentley, Phil Beffrey, and Chandan K. Sen

Introduction

The term functional genomics can be referred to as the "development and application of a global (genome-wide or system-wide) experimental approach to assess gene function by making use of the information and reagents provided by structural genomics."1 It is characterized by high-throughput or large-scale experimental methodologies combined with statistical and computational analysis of the results. The fundamental strategy in a functional genomics approach is to expand the scope of biological investigation from studying single genes or proteins to studying all genes or proteins at once in a systematic fashion. Functional genomics promises to rapidly narrow the gap between sequence and function and to yield new insights into the behavior of biological systems.

As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms be found to assess individual genetic variation. Three methods for obtaining genome-wide mRNA expression data—oligonucleotide "chips,"2 serial analysis of gene expression (SAGE),3 and DNA microarrays4'5—are particularly powerful in the context of knowing the entire genome sequence (and thus all genes).6

Types of DNA Hybridization Arrays

Current array formats can be categorized into the following four groups.

1. Macroarrays: Macroarrays rely on robotically spotted probes that have been immobilized on a membrane-based matrix. The probe density is generally

1 P. Hieter and M. Boguski, Science 278,601 (1997).

2 S. P. Fodor, R. P. Rava, X. C. Huang, A. C. Pease, C. P. Holmes, and C. L. Adams, Nature (London) 364,555 (1993).

3 V. E. Velculescu, L. Zhang, B. Vogelstein, and K. W. Kinzler, Science 270,484 (1995).

4 M. Schena, D. Shalon, R. Heller, A. Chai, P. O. Brown, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 93, 10614(1996).

5 M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, Science 270,467 (1995).

6 V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88,243 (1997).

lower on these arrays compared with those of the other three groups. These arrays mostly use radioactive probe labeling. In some cases chemiluminescent labeling has also been described.

2. Microarrays: Microarrays use a glass or plastic slide as matrix. These arrays have a higher density of probes compared with macroarrays and use fluorescent labeling-based detection.

3. High-density oligonucleotide arrays (gene chips): The probe is generated in situ on the surface of the matrix. The leader in these arrays is Affymetrix (Santa Clara, CA) and their combinatorial synthesis method.

4. Microelectronic arrays: Microelectronic arrays represent one of the more recent formats of hybridization arrays currently under development by Nanogen (San Diego, CA). Instead of a membrane or a glass slide platform, these arrays consist of a set of electrodes covered by a thin layer of agarose coupled with affinity moiety (permitting biotin-avidin immobilization of probes). Selection and adjustment of proper physical parameters enable rapid DNA transport, site-selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity, and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. The incorporation of controllable electric fields gives a new degree of control over probe deposition and target hybridization.7,8

High-Density Oligonucleotide Arrays

The leading arrays in the category of high-density oligonucleotide arrays are manufactured by Affymetrix and utilize the combinatorial synthesis principle.9 The arrays are designed by using a light-directed chemical synthesis process that creates a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps. This process constructs high-density arrays of oligonucleotides. Approximately 20 different probe pairs represent each gene on a chip. Each probe pair consists of a perfect match (PM) oligonucleotide probe and a single-base mismatch (MM) oligonucleotide (Fig. 1). The arrays are designed for gene expression as well as single-nucleotide polymorphism (SNP) detection and they cover a large range of different species. The sequence data that Affymetrix uses to build the arrays are downloaded from public databases such as UniGene and GenBank.

7 C. F. Edman, D. E. Raymond, D. J. Wu, E. Tu, R. G. Sosnowski, W. F. Butler, M. Nerenberg, and M. J. Heller, Nucleic Acids Res. 25,4907 (1997).

8 W. M. Freeman, D. J. Robertson, and K. E. Vrana, Biotechniques 29,1042 (2000).

99618_at (3vs1) IM Intensity: 55 1738

Fig. 1. Approximately 16-20 different probe pairs represent each gene on a chip. Each probe pair consists of a perfect match (PM) oligonucleotide probe and a single-base mismatch (MM) oligonucleotide.

99618_at (3vs1) IM Intensity: 55 1738

Fig. 1. Approximately 16-20 different probe pairs represent each gene on a chip. Each probe pair consists of a perfect match (PM) oligonucleotide probe and a single-base mismatch (MM) oligonucleotide.

For the chips to work properly, a sample must be prepared according to Affymetrix protocols. A brief description of the procedures involved in assessing a gene expression profile, using Affymetix GeneChip arrays, is provided.

Sample Preparation

The oligonucleotides on the chip or microarray are called the probes and the sample (total RNA or mRNA) that is put on to interrogate is called the target. The process is inverted from a traditional Northern analysis.

Total RNA Isolation

RNA is extracted from cells with an RNeasy total RNA isolation kit (Qiagen, Chatsworth, CA). For tissues, RNA is first extracted with TRIzol (Invitrogen, Carlsbad, CA) RNA extraction reagent and then cleaned up with an RNA isolation kit (Qiagen).

cDNA Synthesis

The first strand is synthesized by reverse transcribing the RNA, using the Superscript Choice system (Invitrogen) and oligo(dT)24-anchored T7 primer [high-performance liquid chromatography (HPLC) purified] at 42° for 60 min and then at 70° for 15 min. The second strand is synthesized by using the first-strand synthesis reaction, 5x second-strand buffer, Escherichia coli DNA polymerase, and T4 DNA polymerase. The cDNA is isolated according to the Phase Lock gel extraction (Eppendorf, Hamburg, Germany) procedure.

In Vitro Transcription, cRNA Clean-Up, and Fragmentation

Biotinylated RNA is synthesized with an RNA transcript labeling kit (BioArray High Yield: Enzo Diagnostics, Farmingdale, NY). A detailed protocol is provided with the kit.

In Vitro Transcription Clean-Up. Qiagen RNeasy minicolumns are used to clean up the in vitro transcription (IVT) cRNA. After the clean-up, cRNA is fragmented with 5x fragmentation buffer (200 mM Tris-acetate, pH 8.1; 500 mM potassium acetate; 150 mM magnesium acetate).

GeneChip: Hybridization, Washing, and Scanning

Further sample processing is mostly automated. The hybridization oven 640 automates the hybridization process for GeneChip probe arrays. The oven provides precise temperature control to ensure successful hybridization, and cartridge rotation to provide continuous mixing. Up to 64 arrays can be processed at one time. The GeneChip fluidics station automates the introduction of the nucleic acid target to the probe array cartridge and controls the delivery of reagents and the timing and temperature for hybridization of nucleic acid target to the probe array. Each fluidics station can independently process four arrays at one time. The probe array nucleic acid target is simply loaded on the fluidics station. Information about the type of array to be analyzed is punched in and the software automatically selects the appropriate protocol. Once processing is complete, messages displayed on the PC and the fluidics station indicate that the probe array is ready for scanning. The GeneArray scanner is from Agilent (Palo Alto, CA) and utilizes a charge-coupled device (CCD) camera and an argon ion laser to excite fluorescent molecules incorporated into the nucleic acid target to generate a quantitative hybridization signal (Fig. 2). With precise optics, the GeneArray scanner focuses the laser on 3-//m spots within each of the thousands of probe features contained on the GeneChip probe array. A high-resolution image of the probe array is displayed in real time during scanning, and fluorescence intensity data are automatically stored in a raw file.

0 0

Post a comment