What is gradient PCR?

For a PCR reaction, although there are various PCR primer design softwares or empirical formulas to calculate the optimal annealing temperature, due to the ever-changing combination of bases in the template, the data obtained by empirical formulas may not be "P" for special fragments, and a slight change may have a decisive impact on the results, so "feeling conditions" was once a headache. The appearance of gradient PCR partially solved some problems-the temperature control conditions of each hole can be changed according to the gradient within the specified range during the reaction, and the most suitable reaction conditions can be found in one step according to the results. Not only can the annealing temperature be optimized, but also the denaturation temperature and extension temperature can be optimized-this is very important for the amplification of most high-fidelity Taq enzymes such as Invitrogen, Clontech and Promega, because the optimal reaction temperature of Taq and correction enzyme may be significantly different, so it is very important to optimize the extension temperature. Multiple experiments can be completed on one instrument, which not only saves experimental time, improves efficiency, but also saves experimental cost. For PCR instruments with gradient function, it is necessary to consider the temperature uniformity and accuracy between different gradient tubes in gradient mode, and whether the instrument has the same temperature characteristics in gradient mode and standard mode. This difference may lead to the difference between the optimal conditions obtained in the gradient mode and the results obtained in the standard mode alone. SteadySlope technology is a patent of gradient PCR technology owned by eppendorf, which can reach all the set gradient temperatures at the same temperature change rate, so it has constant temperature in gradient mode. This technology ensures reliable information transmission between gradient mode and normal mode, and will not lead to changes in yield and specificity due to different temperature characteristics. MJ chooses to use different cooling rates in gradient mode without paying patent fees, and the temperature curve between each gradient temperature is different, so switching from gradient mode to normal mode may be problematic. In addition, the gradient PCR instrument using TCT (three-ring) technology adds heating and cooling control areas in gradient PCR mode, which ensures the accuracy of gradient temperature control and makes the temperature uniformity between different gradient tubes better. In-situ PCR amplification can be carried out on the glass slide by adding in-situ PCR module to the PCR instrument. Both MJ and eppendorf PCR instruments provide in-situ adapters to meet different needs. For workers engaged in medical research, it is worthwhile to buy a PCR instrument equipped with a module that supports in-situ PCR. In addition, with the increasing demand for genome Qualcomm quantitative research, various brands have introduced multi-channel Qualcomm quantitative PCR instruments, each with its own specialty: MJ has a kind of 1 host, and has four amplification tanks, each of which can independently control the temperature, and can PCR 96 x4 samples at a time, but once problems occur, all four cannot be used. On the basis of the original 9700, ABI introduced a double 384-hole pedestal, which can complete 384x2 samples at a time, so that the function of the 9700 can be extended to the field of Qualcomm measurement without buying a new machine. Unfortunately, two 384 cans can't control the temperature independently.