DNA arrays are used for a variety of purposes, including sequencing. Large numbers of probes are bound to the chip and hybridization with target DNA occurs on the chip surface.
Earlier DNA technology was largely based on gel electrophoresis, an approach that is both difficult to automate and labor intensive. DNA chips were developed to allow automated side-by-side analysis of multiple DNA sequences. In practice the simultaneous analysis of thousands of DNA sequences is possible. The first chip was introduced by a company called Affymetrix in California in the early 1990's. Since then DNA chips have been used for a variety of purposes including sequencing, detection of mutations and gene expression. DNA chips all rely on hybridization between single-stranded DNA permanently attached to the chip and DNA (or RNA) in solution. Many different DNA molecules are attached to a single chip forming an array of spots on a solid support (the chip).The DNA or RNA to be analyzed must be labeled, usually with fluorescent dyes. Hybridization at each spot is scanned and the signals are analyzed by appropriate software to generate colorful data arrays. Two major variants of the DNA chip exist. Earlier chips mostly used short oligonucleotides. However, it is also possible to attach full length cDNA molecules. Prefabricated cDNA or oligo-nucleotides may be attached to the chip. Alternatively, oligonucleotides may be synthesized directly onto the surface of the chip by a modification of the phosphoramidite method described in Chapter 21. Modern arrays may have 100,000 or more oligo-nucleotides mounted on a single chip.
DNA arrays can detect the presence of multiple small fragments of DNA sequence. A computer then compiles the overall sequence.
The oligonucleotide array detector simultaneously detects and identifies lots of short DNA fragments (i.e., oligonucleotides). It can be used both for diagnostic purposes and for large scale DNA sequencing. The key principle involved is DNA-DNA hybridization (see Ch. 21).
Consider a piece of DNA of unknown sequence. This is denatured to give single strands and one of these is tested for hybridization to a known probe sequence of say, eight bases (an octonucleotide; e.g., CGCGCCCG). If the unknown DNA binds to the probe, then the probe sequence occurs somewhere in the complementary strand of the unknown DNA. The unknown DNA is then tested for hybridization to all other possible stretches of eight bases, one at a time, to see which are found.
oligonucleotide array detector Chip used to simultaneously detect and identify many short DNA fragments by DNA-DNA hybridization. Also known as DNA array or DNA chip
The Oligonucleotide Array Detector 673
FIGURE 24.13 Deducing Sequence From Oligonucleotide Overlaps
By aligning all of the eight-base pair probe sequences that hybridize to the unknown DNA, the computer can determine the sequence of the DNA.
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