RESUMO
Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.
Assuntos
DNA Complementar/metabolismo , Expressão Gênica , Espectrometria de Fluorescência/métodos , Actinas/genética , Algoritmos , Carbocianinas/química , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Corantes Fluorescentes/química , Células HL-60 , Humanos , Células K562 , NF-kappa B/genética , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Fator 1 de Elongação de Peptídeos/genética , Fosfoglicerato Quinase/genética , Rodaminas/química , Proteínas Ribossômicas/genética , Sensibilidade e Especificidade , Espectrometria de Fluorescência/instrumentação , Fator de Transcrição RelA , Tubulina (Proteína)/genética , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent(R) exo-) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent(R) exo-, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.
