The polymerase chain reaction (PCR) is a sensitive method that is commonly used for the isolation, amplification or detection of specific nucleotide sequences in a complex mixture of DNA, like genomic DNA. However, the sequences of the ends of the target region must be known. PCR has numerous applications in basic and clinical research, human genetics testing, and forensics. It is a kind of in vitro
DNA replication, which is repeated many times for a particular DNA segment only. Typically, two synthetic oligonucleotides complementary to the 3’ ends of the target DNA region are provided, after binding specifically to the denatured DNA target, the oligonucleotides are used as primers by a temperature-resistant DNA polymerase to synthesize the complementary strand. At each round of replication, the number of copies of the DNA between the primer sites is doubled. Hence, repeated cycles of denaturation, annealing and extension result in the exponential amplification of the target DNA.
Every PCR reaction, (i.e. each set of primers) needs to be optimized. Magnesium ion (Mg2+ ) concentration and annealing temperature (Ta) are 2 parameters, which are of particular importance when optimizing PCR. The optimal Ta depends on the sequence of the primers (%GC and length) and their homology to the template DNA. As a rule of thumb, the Ta is 4ºC to 10ºC lower from the average melting temperature (Tm) of primers. However, this calculation does not necessarily result in the optimal Ta and hence Ta has to be determined empirically. For this reason, many independent PCR reactions have to be run with various Tas. Gradient PCR makes running these reactions convenient by carrying out many PCR reactions at the same time with different Tas. This reduces the amount of time required to select the optimal Ta, where the yield of desired product is high and non-specific products are absent, or at least minimized. Even when running reactions with several Mg2+ concentrations and various Tas, it is possible to optimize a PCR reaction conditions in one run when using a gradient thermal cycler.
The Gradient Palm–Cycler™ from Corbett is a user-friendly gradient thermal cycler; it is available in a 96-well or 384-well format. Individual 0.2 ml tubes or strip tubes can be used. The temperature range of operation is from 4ºC to 99ºC with 8 Peltier devices engaged in fast heating and cooling. Maximum heating and cooling rate is 2.5ºC/sec for the entire block with 1.5ºC/sec as the average heating/cooling rate experienced by an individual tube. The Palm-Cycler™ has a heated top plate that eliminates the need for oil overlay on the reactions. The thermal cycler is under the control of Palm-Cycler™ software installed on an integrated HP computer, the palm PC. The palm PC is locked onto the thermal cycler and can only be removed with the special Allen-key provided, ensuring security of the palm PC. The advantage of the palm PC is that we have all the benefits of a computer: For example, Internet connection, which enables the easy software updates. OLIGO CALC Software aids in calculating molecular weight, Tm and potential self-hybridization of an oligonucleotide.
In addition, a touchdown cycling feature is provided; this allows the Ta to be reduced by 1ºC per cycle. Basically, in Touchdown PCR, a Ta that is higher than the optimal Ta is employed in early PCR cycles until a 'touchdown' annealing temperature is reached, which is then used for the remaining number of cycles. This allows for the enrichment of the correct product over any non-specific product. This option avoids the lengthy programming, as is often necessary with other thermal cyclers. Similarly, a long-range parameter allows cycle lengths to be changed in designated increments after a particular cycle number is reached.
PCR is a routine technique in our lab for experiments like cloning, methylation analysis, chromatin immuno-precipitation assay and RT-PCR. The only problem we have with this instrument is that 0.5 ml tubes cannot be used and so large PCR reaction volumes have to be distributed to 0.2 ml tubes. Also, if the machine is not in regular use then the palm PC needs to be charged before use.
Hence, in my view, Corbett’s Gradient Palm-Cycler™ is a must for any molecular biology lab and is highly recommended.