The Single-Tube PCR Kit from Takara allows nucleic acid extraction and the subsequent PCR to occur in a single tube (0.5ml) in a short amount of time. It can be used with small volumes (3 to 10ul) of whole blood from human, mouse or rat. It uses a convenient format for a highly efficient and specific PCR reaction. The kit contains optimized components that allow both extraction and PCR amplification to take place in what is referred to as a “single-tube PCR”. It cannot be used only for extraction.
The kit components of the Single-Tube PCR Kit are Gene Trap Solution which has been aliquoted into 0.5ml PCR tubes, Washing Solution, 2X PCR Solution and Master Mix. Primers are not provided with the kit. The 2X PCR Solution is composed of TaKaRa ExTaq, reaction buffer, and dNTP mixture. TaKaRa ExTaq is the key enzyme for LA (long amplification) PCR technology; this Taq provides more efficient amplification and higher fidelity than conventional Taq DNA polymerase.
The protocol requires that the Gene Trap Solution and Washing Solution must be pre-equilibrated to room temperature. The protocol is as follows: Add 10 ul of blood sample into the 0.5 ml tubes supplied with Gene Trap Solution and leave at room temperature for 10 minutes, centrifuge at 8000 g for 5 minutes. Remove the supernatant and add 0.3 ml of washing solution and mix by inverting the tube, centrifuge again at 8000 g, this time for 2 minutes. Gently remove the supernatant with a micropipette; the pellet is the DNA, ready for amplification. Into the tube containing the precipitate, add 50 ul of 2x PCR Solution and 10-100 pmol each of forward and reverse primers. Adjust the total volume to 100 µl with sterilized distilled water. After thorough mixing, the tubes are ready for amplification. The tubes are placed in the thermal cycler. After the pretreatment at 94ºC for 20 minutes to ensure denaturation of the DNA, thermal cycling is started. This kit enables detection a single copy gene after 25-30 cycles.
We checked the kit by extracting DNA from human blood (10 µl) and amplifying with β-globin primers and obtained very good results (product size was 408 bp) as there was a clear band with no smearing and no primer dimer. We have also tried this kit using a smaller volume of blood (5 µl); to do so, we increased the number of PCR cycles to 30 and we still obtained a single, clear PCR product.
I don’t think there is another cheaper option for DNA extraction and amplification in a single tube, in a very quick manner. The main limitation of this kit is that only blood can be used.
Tarun Jain
Application Specialist
Biotron Healthcare
Molecular Biology Laboratory