The emerging science of genomics must deal with two major quantitative realities. First, there are a large number of genes in eukaryotic genomes. Second, the pattern of gene expression in different tissues at different times is quite distinctive. For example, a skin cancer cell at its early stage may have a unique mRNA "fingerprint" that differs from that of both normal skin cells and the cells of a more advanced skin cancer.
To find these patterns, scientists could isolate the mRNA from a cell and test it by hybridization with each gene in the genome, one gene at a time. But it would be far simpler to do these hybridizations all in one step. To facilitate this, one needs some way to arrange all the genes in a genome in an array on some solid support.
DNA chip technology provides these large arrays of sequences for hybridization. DNA chips were developed by modifying methods that have been used for decades in the semiconductor industry. You may be familiar with the silicon microchip, in which an array of microscopic electric circuits is etched onto a tiny chip. In the same way, DNA chips are glass slides to which a series of DNA sequences are attached in a precise order (Figure 16.10). Typically, the slide is divided into 24 x 24 |M squares, each of which contains about 10 million copies of a particular sequence up to 20 nucleotides long. A computer controls the addition of nucleotides in a predetermined pattern. Each 20-base-long sequence hybridizes to only one genomic DNA (or cDNA) sequence, and thus is a unique identifier of a gene. Up to 60,000 different sequences can be placed on a single chip.
If cellular mRNA is to be analyzed, it is usually incubated with reverse transcriptase (RT) to make cDNA (see Figure 16.8), and the cDNA is amplified by the polymerase chain reaction (PCR) prior to hybridization (see Figure 11.20). This technique is called RT-PCR, and it ensures that mRNA sequences naturally present in only a few copies (or in a small sample, such as a cancer biopsy) will be numerous enough to form a signal. The amplified cDNAs are coupled to a fluorescent dye and used to probe the DNA on a chip. Those cDNA sequences that form hybrids can be located by a sensitive scanner. With the number of genes that can be placed on a chip approaching that of the largest genomes, DNA chips will result in an information explosion on mRNA transcription patterns in cells in different physiological states.
Another use for DNA chips is in detecting genetic variants. Suppose one wants to find out if a particular gene, which is 5,500 base pairs long, has any mutations in a particular individual. One way would be to sequence the entire gene, but that would be expensive and time-consuming. On the other hand, DNA chip technology can be used to make 20-nucleotide fragments including the entire gene and all (or nearly all) of its known point mutations and small deletions. Then, hybridization with the individual's DNA might reveal a particular mutation if it hybridized to a mutant sequence on the chip. This rapidly developing technology could be an important step toward individualized diagnosis and therapy for human genetic diseases.
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