Nucleic acid capture assay, a new method for direct quantitation of nucleic acids
| Autor: | Grace Mausisa, Anne Wong, Mark Vasser, Jean‐Philippe Stephan, Wai Lee T. Wong, Pamela Chan, Mogens Jakobsen, Parkash Jhurani, Siao Ping Tsai, Elaine Mai |
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| Rok vydání: | 2003 |
| Předmět: |
chemistry.chemical_classification
Nucleic acid quantitation Reverse Transcriptase Polymerase Chain Reaction Branched DNA Signal Amplification Assay RNA Gene Expression Reproducibility of Results Biology Sensitivity and Specificity Chemistry Techniques Analytical chemistry.chemical_compound chemistry Biochemistry Nucleic Acids Genetics Nucleic acid Humans Nucleotide DNA microarray K562 Cells Linker DNA Fetal Hemoglobin NAR Methods Online |
| Zdroj: | Nucleic acids research. 31(6) |
| ISSN: | 1362-4962 |
| Popis: | Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications. |
| Databáze: | OpenAIRE |
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