Jacqueline A. M. Vet

Design and optimization of molecular beacon real-time polymerase chain reaction assays.

During the last few years, several innovative technologies have become available for performing sensitive and accurate genetic analyses. These techniques use fluorescent detection strategies in combination with nucleic acid amplification protocols. Most commonly used is the real-time polymerase chain reaction (PCR). To achieve the maximum potential of a real-time PCR assay, several parameters must be evaluated and optimized independently. This chapter describes the different steps necessary for establishing a molecular beacon real-time PCR assay: (1) target design, (2) primer design, (3) optimization of the amplification reaction conditions using SYBR Green, (4) molecular beacon design, and (5) molecular beacon synthesis and characterization. The last section provides an example of a multiplex quantitative real-time PCR.

Molecular beacons: colorful analysis of nucleic acids.

The completion of the Humane Genome Project has resulted in an exponential rise in the demand for molecular diagnostic assays. To meet this demand, several innovative technologies have become available for performing homogeneous genetic analyses. For this type of assay, special detector probes are necessary. In 1996, Tyagi and Kramer described fluorogenic hairpin-shaped detector probes, called ‘molecular beacons’, which are extraordinarily specific. Since they characterize alleles by the generation of fluorescent signals, they are perfectly suited for homogeneous genetic analysis. Molecular beacons assays are simple, fast, inexpensive, sensitive, utilize a high-throughput format, enable the testing of many samples simultaneously and allow the detection of a series of different agents in the same assay tube. This review is designed to give the reader a greater understanding of the exciting applications of molecular beacons in DNA, RNA and protein studies.

Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler.

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low‐throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi‐automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 μl of EDTA‐treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi‐automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi‐automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.

Molecular Beacons: Hybridization Probes for Detection of Nucleic Acids in Homogeneous Solutions

Molecular beacons are oligonucleotide probes that can report the presence of specific nucleic acids in homogeneous solutions (Tyagi and Kramer, 1996). They are usefulin situationswhere it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes, such as in real-time monitoring of polymerase chain reactions in sealed tubes or in detection of RNAs within living cells. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid (Figure 1).They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence which can be detected.