Nucleic acid capture assay, a new method for direct quantitation of nucleic acids.
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 In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding.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.
Tsai
Siao Ping
SP
Assay and Automation Technology Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Wong
Anne
A
Mai
Elaine
E
Chan
Pamela
P
Mausisa
Grace
G
Vasser
Mark
M
Jhurani
Parkash
P
Jakobsen
Mogens H
MH
Wong
Wai Lee T
WL
Stephan
Jean-Philippe
JP
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005319:Fetal Hemoglobin / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D020014:K562 Cells; D009696:Nucleic Acids / Q000032:analysis* / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D009696:Nucleic Acids; D012313:RNA; D005319:Fetal Hemoglobin