Typing of consensus polymerase chain reaction productsendpoint measurement

Typing of PCR products was traditionally performed by means of dot blotting or Southern blotting and hybridization with type-specific oligonucleotides. More recently, reverse hybridization techniques were introduced. These methods rely on the hybridization of labeled consensus

PCR products to HPV type-specific oligos immobilized on filters. Examples are reverse line blot (RLB) analysis following MY09/11[7] or GP5+/6+[6] consensus PCR or a line probe assay (LiPa) following SPF PCR.[10] Detection of the hybridized PCR product is performed by a colorimetric reaction[7,10] or by chemoluminescence,[6] the latter allowing repeated usage of the same filter.[6] Instead of filters, glass microarrays of HPV type-specific probes can also be used.[11] A schematic representation of the principle of reverse hybridization is given in Fig. 1. These methods are far less laborious than normal hybridization methods when it comes to detection

Fig. 1 Reverse hybridization methods: RLB.[6] Schematic representation of the principle. HPV type-specific probes are attached to a membrane using a miniblotter with slits. Subsequently, the membrane is rotated 90° in the miniblotter. Consensus PCR products are generated using hapten-labeled primers. After denaturation, the single-stranded PCR products are pipetted into the slits of the miniblotter. If a particular HPV type is present in a sample, hybridization will occur on the crossing point of the sample slit with the respective HPV probe line. Subsequently, hybridization is detected using a labeled antibody directed against the hapten and visualized using chemoluminescence.

Fig. 1 Reverse hybridization methods: RLB.[6] Schematic representation of the principle. HPV type-specific probes are attached to a membrane using a miniblotter with slits. Subsequently, the membrane is rotated 90° in the miniblotter. Consensus PCR products are generated using hapten-labeled primers. After denaturation, the single-stranded PCR products are pipetted into the slits of the miniblotter. If a particular HPV type is present in a sample, hybridization will occur on the crossing point of the sample slit with the respective HPV probe line. Subsequently, hybridization is detected using a labeled antibody directed against the hapten and visualized using chemoluminescence.

of multiple HPV infections. For group-specific detection of HPVs without high-resolution typing, an enzyme immunoassay (EIA) can be applied conveniently using cocktails of, for example, HR- or LR-HPV probes.[12]

Two nonhybridization typing methods following consensus PCR are sequence analysis of the PCR product[13'M] and restriction fragment length polymorphism (RFLP) analysis.1-15-1 RFLP implies the digestion of consensus PCR products with restriction endonucleases and comparison of the digestion pattern with those of known HPV types. These techniques are useful if unknown types of HPV are present in the specimens, but they have several drawbacks as compared with hybridization methods. For example, RFLP and sequence analysis are not suitable for the detection of infections with multiple HPV types: these will usually give an uninterpretable mix-up of digestion/sequence patterns. In addition, these two methods are less suitable for high-throughput analyses because they are relatively laborious. Finally, RFLP and sequence analysis are less sensitive than hybridization methods because more PCR product is needed to generate a positive signal.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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