Analysis of Nucleic Acids by Tandem Hybridization on Oligonucleotide Microarrays

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: 1. The need to generate single-stranded target nucleic acids in order to achieve optimal hybridization signals. 2. Spontaneous formation of secondary structure in the single-stranded target nucleic acid, causing certain stretches of target sequence to be poorly accessible to hybridization. 3. Imperfect specificity of hybridization, making it difficult or impossible to distinguish between certain sequence variations. 4. 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. 5. 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. 6. 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.