The DNA microarray is one of more recent promising techniques for mutational analysis. These devices, in their simplest form, exploit direct hybridization. They quickly occupied niches in nearly every area of nucleic acid analysis as several reviews indicate.74_76 Once hybridization patterns or ''signatures'' of large numbers of mutant alleles of interest are known, mounting a search for those signatures in many different samples is possible. On the other hand, screening for all possible new mutations with microarrays is more difficult than searching for known mutations.77
Several factors may contribute to the potential difficulty in identification: (1) The microarray-based system is more sensitive to detection of homozygous base changes than heterozygous changes. Although it is possible to use micro-arrays for heterozygous screening,78 for general use in the detection of heterozygous base changes, accuracy must be improved. It is not yet ready for applications needing >98% heterozygous mutation detection. (2) Variations in target sequence composition, including the presence of repetitive sequence elements, can exert a major influence on the sensitivity of the system, and may lead to unacceptably high false-negative rates. (3) Intramolecular and intermolecular structures, such as hairpins and G-quartets, present in either target or probe can make hybridization less predictable. Variant sequences that disrupt secondary structures may act to increase or decrease the affinity to ''perfect match'' probes. Deletions and insertions can pose special problems because they may form duplexes containing bulged nucleotides with a wild-type probe that in turn can alter the predictability of the outcome. Unfortunately, few systematic studies have been conducted to determine what steps must be taken to reduce or circumvent the effects of these factors.
The discriminating power that can be achieved by direct hybridization alone can be improved by coupling target hybridization with enzymatic primer extension with DNA polymerase.77,79 DNA polymerase uses oligonucleotides tethered to the membrane surface, or in a microtiter dish, via the 5'-end linkage to leave exposed the free 3'-OH group. Chain-terminating, labeled dideoxynucleotide triphosphates (ddNTPs) are used in primer extension reactions in which the hybridized target and oligonucleotide probes serve as template and primer, respectively. The enzyme incorporates and extends the primer by only one base, which is complementary to the next base in the target. The enzyme drives the reaction further with greater specificity than is possible with direct hybridization alone under conditions of high stringency. This process is known as ''minisequen-cing''80or ''genetic bit analysis.''81 Methods similar in principle that use DNA ligase instead of polymerase have been adapted for use with microarrays. A mixture of all four ddNTPs, each labeled with a different fluorescent dye, can be used in the primer extension reactions, and the identity of the extended ddNTP-labeled product is determined by fluorescent microscopy. It should be noted that commercially available Affymetrix arrays tethered to the surface through 3'-end linkages are unsuitable for this task.
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