Analysis of Nucleic Acids by Tandem Hybridization on Oligonucleotide Microarrays
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Oligonucleotide arrays (also known as DNA chips, gene chips, or genosensor chips) are emerging as a powerful research tool in nucleic acid sequence analysis. Several technical challenges remain to be solved, however, before oligonucleotide arrays can reach their full potential and be implemented in a robust fashion. Some of these challenges are as follows:
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The need to generate single-stranded target nucleic acids in order to achieve optimal hybridization signals.
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Spontaneous formation of secondary structure in the single-stranded target nucleic acid, causing certain stretches of target sequence to be poorly accessible to hybridization.
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Imperfect specificity of hybridization, making it difficult or impossible to distinguish between certain sequence variations.
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The strong influence of base composition on the stability of short duplex structures, making it difficult to use an extensive array of oligonucleotides (differing in base composition) to analyze a nucleic acid sample under a single hybridization condition.
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Multiple occurrence of sequences complementary to short oligonucleotide probes within the nucleic acid sample, limiting the genetic complexity of a nucleic acid sample that can be analyzed by arrays of short oligonucleotide probes.
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The need to label each nucleic acid analyte prior to hybridization to the DNA probe array, a significant factor in the overall time and cost of analysis.
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