Single-tube two-round real-time PCR was achieved in LightCycler capillaries by separating the first-round PCR mixture from the second-round PCR mixture by a layer of silicone oil placed in between for the duration of the first round of PCR. Thus the first round of PCR was exclusively performed with the first-round primers and reagents. Then, after the completion of the first-round PCR a brief centrifugation step forced the second-round reagents including the second-round primers to move underneath the silicone oil. Thus second-round reagents were united with first-round products and the second-round PCR was performed (Fig. 1). As the capillaries remained closed during the entire procedure, product carryover into samples analyzed in parallel was avoided between both rounds of PCR.
The feasibility of this method was initially demonstrated by using nested pairs of primers for the PCR-mediated detection of the MBR/JH chromosomal translocation t(14; 18) (q32;q21) in cells of the human B-lymphoma cell line DoHH2. As those cells carry this translocation, they were used as source for MBR/JH-specific DNA. The first-round and second-round PCR mixtures were prepared with equal volumes of 15 pL that were separated in the LightCycler capillary by 5 pL of silicone oil. Both PCR mixtures contained SYBR Green I as detecting fluorochrome. First-round PCR primers were used at about half the concentrations more than were used for the second-round primers. The second-round mixture was void of Taq polymerase and contained only primers and buffer. Capillaries were loaded with the first-round PCR mixture followed by the silicone oil. A brief centrifugation (735 xg for 5 sec) established the two separate phases with the silicone oil on top, onto which the second-round PCR mixture was layered (Fig. 1). The capillaries were closed, and the first and second PCR cycling programs were performed interrupted only by the centrifugation step after the first round of PCR. Product generation was recorded during both rounds of PCR, and after the completion of the second round of PCR melting curve analysis was performed to discriminate MBR/JH-derived products from primer dimers (Fig. 2). The single-tube two-round PCR detected the MBR/JH fusion gene from as low as 50 pg DoHH2 cell-derived DNA (Fig. 2A-D). This detection limit corresponded to about two to three cells per PCR. For comparison, one-round PCR using the first-round primers only detected the MBR/ JH fusion gene from 500 pg DoHH2 DNA. This corresponded to a detection limit of about 20 to 30 cells per PCR only. Thus, the two-round PCR was found to exhibit an approximately 10-fold increased sensitivity than the one-round PCR with this application.
As mentioned above, the described principle of singletube two-round real-time PCR relates to the physical separation of the first-round PCR mixture from the second-round reagents during the first round of PCR. To test whether the 5 pL of silicone oil sufficed for the effective separation of both reaction mixtures, mock single-tube two-round real-time PCRs were performed with first-round mixtures, prepared without primers. Instead, the primers were placed on top of the oil layer. After completion of the mock first round of temperature cycling, centrifugation was performed and the second round of temperature cycling was carried out. Thus, in repeated experiments amplification products were obtained only in the second round of temperature cycling, and not during the first round (Fig. 3).
Furthermore, when first- and second-round reaction mixtures are unified after the completion of first-round PCR, both mixtures should mix up to homogeneity. In order to accomplish this, both mixtures were prepared to equal volumes. However, mixing experiments with bromphenol blue as indicator dye in the second-round mixture revealed that homogeneity of both mixtures was not obtained immediately after centrifugation. Instead, the centrifugation forced the upper liquid phase to move underneath the first-round mixture. When the temperature
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The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.