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Unformatted text preview: Toward Improved Biochips Based on Rolling Circle AmplificationInfluences of the Microenvironment on the Fluorescence Properties of Labeled DNA Oligonucleotides E LKE M AYER-E NTHART , a J ULIEN S IALELLI , a K NUT R URACK , a U TE R ESCH-G ENGER , a D ANIELA K OSTER , b AND H ARALD S EITZ b a Federal Institute for Materials Research and Testing (BAM), Div. I.5, Berlin, Germany b Max Planck Institute for Molecular Genetics, Vertebrate Genomics, Berlin, Germany Microarrays have become an increasingly important tool for biotechnology and molecular diag- nostics. Despite many advantages, their sensitivity is still insufficient for such tasks as the analysis of small sample quantities and for the detection of alterations in gene expression of low-abundance genes. Accordingly, amplification strategies are necessary. Approaches to amplify the signal inten- sity include the increase of the number of dye molecules per target through either particle labels or rolling circle amplification, as used for this study. Key words: rolling circle amplification; fluorescence; biochips; microarrays; signal amplification; Cy3-labeled oligonucleotides; DNA technology Introduction The use of microarray technology in biological and clinical applications is constantly increasing. The technique has a unique potential for the analysis of many different samples in parallel. 14 Several kinds of biochips are available according to the biomolecules (nucleic acids, proteins, antibodies, and oligosaccha- rides, as well as small chemical compounds) that are immobilized on the chip surface. 5 In DNA microarray technology, DNA fragments and oligonucleotides are immobilized or directly synthesized on a slide. 3 In prin- ciple, these chips are used for expression profiling of tis- sues and cell cultures. The development of specialized DNA biochips for future use in medical diagnostics is already in progress. 6 Methods that are currently estab- lished typically need 1520 g of mRNA as starting material. However, only a few nanograms of nucleic acids can usually be extracted because, for instance, of the few cells obtained from biopsy samples. Also, iso- lation of RNA from RNA-poor tissues such as ocular epithelium or fatty tissue is difficult. Hence, consider- Address for correspondence: Dr. Elke Mayer-Enthart, Federal Institute for Materials Research and Testing (BAM), I.5 Bioanalytics, Richard- Willstatter-Str. 11, 12489 Berlin-Adlershof, Germany. Voice: + 030-8104- 5961; fax: + 030-8104-5005. firstname.lastname@example.org able efforts have been made to reduce the detection limit of conventional microarray analysis. Signal Amplification by Analyte Multiplication There are two strategies to improve the sensitivity of biochips: (1) preamplification methods to increase the number of analyte molecules and (2) signal amplifica- tion strategies....
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