Reduced PCR runtimes and increased yields
Kerstin Isermann, Arora Phang, Eppendorf SE
PCR is one of the most popular methods in laboratories across the world and is routinely used in research, reference and clinical fields for various applications. Over the years, PCR technology has evolved with the needs of scientists; including developments like real-time PCR and digital PCR.
As time is one of the most precious factors in a laboratory, a decrease in amplification time and thus higher throughput are advantageous for every lab. A lot of efforts have been put into speeding up PCR reactions.
One approach is the establishment of two-step thermal cycling protocols. Other developments include thermal cyclers with higher ramp rates and special kits containing polymerases with faster extension rates. However, a significant reduction in cycle times often requires specialized equipment and adapted procedures that may not be easily implemented in a standard lab.
Therefore, typical standard end-point PCR protocols still take about an hour or more. One reason fast or rapid PCR protocols are not widely-used yet is that the transfer of an established PCR protocol to a fast protocol often compromises PCR yield and specificity. Furthermore, rapid protocols are often linked to very low reaction volumes (< 5 μL) and these are rarely applicable for standard applications. The use of special fast PCR master mixes does reduce runtimes but their full potential can only be realized in combination with fast thermal cyclers.
The limiting factor here is the speed of heat transfer from the block to the sample. In the past, this limitation was mainly addressed by making the vessel walls thinner.