生物化学技术

Real-time TaqMan� polymerase chain reaction (PCR) assays allow quantification of the initial amount of target in a specimen, specifically, and reproducibly. The major limitation of TaqMan PCR assays is that they do not detect the size of the amplified target sequence. TaqMan PCR coupled with ...

A novel closed-tube format telomeric repeat amplification protocol specifically adapted to real-time detection and quantification of telomerase activity was developed. The assay utilizes energy transfer primers, which emit fluorescence only upon incorporation into pol ...

Multiplex quantitative polymerase chain reaction (PCR) based on novel design of fluorescent primers is described. Self-quenched fluorogenic primers are labeled with a single fluorophore on a base close to the 3′-end with no quencher required. A tail of 5-7 nucleotides is added to the 5′-end of t ...

Application of a real-time detection system based on a novel primer design in gene expression profiling is described. In this system, called LUXTM (Light Upon eXtension), the generation of signal is based on a single fluorescent dye molecule that is attached to an oligonucleotide close to the 3′- ...

Mitochondrial respiratory chain disorders are a group of clinically and genetically heterogeneous diseases. Several mitochondrial (mt)DNA point mutations are responsible for common mitochondrial diseases. These pathogenic mtDNA point mutations are usually heterop ...

The Invader� assay (Third Wave Technologies) is a homogeneous, isothermal DNA probe-based method for sensitive detection of nucleic acid sequences. Invader reactions are performed directly on genomic DNA or total RNA targets; however, polymerase chain reaction- or reverse transcr ...

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting fluorescence energy transfer and combinatorial synthesis to generate a large number of unique fluorescence emission signatures from a limited number of fluorophores, allow multiple biol ...

The Amplifluor method for single-nucleotide polymorphisms (SNP) genotyping provides homogeneous assays that utilize a pair of universal energy transfer-labeled primers. The main advantage of this single-step, loci-independent, low-cost method is that it can be readily adapt ...

Fluorescence resonance energy transfer (FRET) provides distance information between a donor and an acceptor dye in the range of 10-100 �. Knowledge of the exact positions of some dyes (e.g., fluorescein, rhodamine, or Cy3) with respect to nucleic acids and DNA design enables us to translate these ...

Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure and dynamics of nucleic acids in solution. Such a technique often uses only one donor fluorophore and one acceptor fluorophore to probe the distance and its changes between the two labeled sites. To ...

Biosensors are devices that amplify signals generated from the specific interaction between a receptor and an analyte of interest. RNA structural motifs called aptamers have recently been discovered as receptor components for biosensors owing to the ease with which they can be evolved ...

A guanine (G)-quadruplex DNA motif has recently emerged as a biologically important structure that is believed to interfere with telomere maintenance by telomerase.G-quadruplexes exhibit four-stranded structures containing one or more nucleic acid strands with central cha ...

Recent development of solution-phase molecular-scale Boolean calculations using deoxyribozymes is potentially an important step toward the development of autonomous therapeutic and diagnostic devices. Here, the construction of basic YES, AND, ANDNOT, and ANDANDNOT deox ...

The catalytic RNA subunit of Escherichia coli RNase P (M1 RNA) is a structure-specific endonuclease that cleaves pre-tRNA substrates in trans. By combining catalytic and substrate RNAs in one RNA molecule, self-cleaving ribozymes have been created (1–3). These constructs were convert ...

To understand the function of a ribozyme, it is necessary to decipher the reaction the catalytic RNA promotes. As more and more RNA catalysts are discovered, then activity will be compared to gain greater understanding of how each functions. Measurement of reaction parameters provides vital ...

In determining reaction rates, catalytic RNA molecules follow the same rules as protein enzymes. These RNA enzymes or ribozymes use similar processes to promote their function as do their protein counterparts, such as binding their substrates in order to make bimolecular reactions uni ...

Ribozymes have been successfully designed to downregulate gene expression in vivo. To enhance the probability of success in vivo, the investigator should have available the ribozyme with the highest catalytic activity feasible. This can only be determined by in vitro assays for cataly ...

Hammerhead ribozymes, in their trans-cleaving form catalyze the cleavage of complementary RNA strands, which contain the UX sequence (where X is A, U, or C) Immediately 5′ to the site of cleavage, although not all such sequences are cleaved with the same efficiency (1,2). The reactivity of the hammer ...

Ribozymes are strands of RNA that are able to catalyze reactions. The ability to quantify accurately the catalytic activity of ribozymes is central to the process of defining their mechanisms of action. The catalyzed reaction is most commonly a trans-esterification reaction, resulting ...

Small catalytic RNA motifs, such as the hammerhead or hairpin ribozyme, have been used to design artificial nucleases that can cleave a selected target RNA sequence (1–3). This was first demonstrated by Uhlenbeck in which the catalytic core of a hammerhead motif was combined with antisense fla ...