Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes

Publisher Summary The real-time monitoring of polymerase chain reaction (PCR) amplifications, or kinetic PCR analysis, allows one to follow PCR DNA replication on a cycle-by-cycle basis. In contrast with other ways of quantitation based on PCR, kinetic PCR has the advantages of being a "closed-tube" assay, including mechanical simplicity, high throughput, and containment of potential carryover contamination. It also has the ability to quantify over a range of many logs of target concentration without the sample dilution needed by other methods. This chapter discusses the various approaches to monitor the PCR amplifications including cycle-by-cycle monitoring. Cycle-by-cycle monitoring allows one to trace a "growth curve" or profile for a PCR that is perfectly analogous to that of a bacterial culture's growth. The chapter empathizes on the use of a charged-coupled device (CCD) camera, which is a simpler, less expensive method of monitoring multiple PCRs. It does the quantitative, digital imaging of the PCRs as they sit in the thermocycler block. A thermocycler block is a compact, light-tight enclosure, fitted over a thermal cycler, avoids the need for a darkroom. It requires less space, and decreases the distance between the camera and the samples, making the collection of fluorescence more efficient. The chapter illustrates several figures and diagrams for the better understanding of the machines.