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Fig. 1 Schematic sketch of single-tube two-round real-time PCR in glass capillaries on the LightCycler instrument. (View this art in color at www.dekker.com.)

Fig. 2 Example of single-tube two-round real-time PCR for the detection of MBR/JH translocation. Real-time PCR on the LightCycler instrument was performed with graded amounts of genomic DNA (50 to 0.01 ng). Product generation was recorded in real-time fashion during the first round (A) and the second round (B) of PCR. Note that baseline normalization was switched off to show the increase of fluorescence during both rounds of the two-round PCR in comparable fashion. (C) After the amplification melting curve analysis was performed. The products specific for the MBR/JH fusion gene generated peaks corresponding to a melting temperature of about 89.5°C. As the melting peaks superimposed upon each other, only parts of the results are presented for clarity of the figure. (D) The amplification products after both rounds of PCR were resolved by standard agarose gel electrophoresis (3% agarose, 100 V for 1 hr, visualization by ethidium bromide staining and UV illumination). The first-round primers generated a 199-bp product whereas the second-round primers generated a 178-bp product. The nucleic acid molecular weight marker was resolved in lane M. (From Ref. [9].)

Fig. 2 Example of single-tube two-round real-time PCR for the detection of MBR/JH translocation. Real-time PCR on the LightCycler instrument was performed with graded amounts of genomic DNA (50 to 0.01 ng). Product generation was recorded in real-time fashion during the first round (A) and the second round (B) of PCR. Note that baseline normalization was switched off to show the increase of fluorescence during both rounds of the two-round PCR in comparable fashion. (C) After the amplification melting curve analysis was performed. The products specific for the MBR/JH fusion gene generated peaks corresponding to a melting temperature of about 89.5°C. As the melting peaks superimposed upon each other, only parts of the results are presented for clarity of the figure. (D) The amplification products after both rounds of PCR were resolved by standard agarose gel electrophoresis (3% agarose, 100 V for 1 hr, visualization by ethidium bromide staining and UV illumination). The first-round primers generated a 199-bp product whereas the second-round primers generated a 178-bp product. The nucleic acid molecular weight marker was resolved in lane M. (From Ref. [9].)

cycling was initiated, both mixtures were found mixed to homogeneity after about four to five temperature cycles. When much smaller volumes such as 2 or 5 pL for the second-round mixtures were used compared with the first-round mixtures, homogeneous mixing of both phases was not observed in experiments with bromphenol blue, and nested primer PCR did not occur either.[9]

In a further development of single-tube two-round realtime PCR reverse transcription (RT) was tried to perform within the same closed reaction tube used for the two-round PCR. As detection format, FRET hybridization probes were used instead of SYBR Green I. In this application a two-tube nested RT real-time PCR on the LightCycler instrument was merged into one capillary.[12] The first-round reaction mixture contained the reagents for the RT and for the first round of PCR. The primers for the first and second round of PCR were chosen to exhibit marked differences in their annealing temperatures (8-10°C). So the selected high annealing temperature of the first-round primers resulted in high specificity in the first round of PCR. As a lower annealing temperature was applied to the second-round PCR, potential spurious products of the first-round PCR were further amplified to arrive at a high sensitivity for the assay. In order to further increase sensitivity, the second-round PCR mixture was complemented with a hot start Taq polymerase. This enzyme needs extensive heating (about 7-10 min at 95°C) to be rendered active. As this was not reached with the temperature profile applied to the first round of PCR, the formation of primer dimers did not occur in the second-round mixture during the first round of PCR. As a result the developed HCV-specific assay exhibited an about 20-fold increased detection limit compared to a single-round HCV-specific real-time RT-PCR assay. With the

10 15 20 25 Cycle Number

10 15 20 25 Cycle Number

Fig. 3 Effectiveness of the oil layer for single tube two-round real-time PCR. (A) First-round PCRs in the presence of SYBR Green I were performed with primers (DNA+primer), with primers layered on top of the oil phase (DNA+oil+primer), and without primers (DNA+oil — primer) and with H2O instead of DNA, with primers (H2O+primers) and with primers layered on top of the oil phase (H2O +oil+primers). Fluorescence intensities were recorded in real-time fashion. After the first-round PCR the capillaries were spun down and second-round PCR was performed with real-time recording of the fluorescence intensities. (From Ref. [11].)

10 15 20 25 Cycle Number

Fig. 3 Effectiveness of the oil layer for single tube two-round real-time PCR. (A) First-round PCRs in the presence of SYBR Green I were performed with primers (DNA+primer), with primers layered on top of the oil phase (DNA+oil+primer), and without primers (DNA+oil — primer) and with H2O instead of DNA, with primers (H2O+primers) and with primers layered on top of the oil phase (H2O +oil+primers). Fluorescence intensities were recorded in real-time fashion. After the first-round PCR the capillaries were spun down and second-round PCR was performed with real-time recording of the fluorescence intensities. (From Ref. [11].)

detection limit of 100 IU/mL this single-tube two-round RT-PCR assay was found suitable for the routine testing of patient samples and for the routine screening of donor samples for the preparation of blood-derived products.[12] The following limitations were, however, observed with this HCV-specific single-tube two-round real-time RT-PCR approach. The first-round PCR was performed in the absence of the FRET hybridization probes. When they were already added to the first round of PCR, the detection limit was found lower than without hybridization probes. As the probes were omitted in the first-round reaction mixture, quantification was not possible during the first round of PCR, only during the second round of PCR to which they were applied. Therefore this procedure caused a limited range of quantification from about 2500 IU/mL to about 100 IU/mL only. This is in marked contrast to the ranges of quantification with one-round real-time PCR, which usually covers several orders of magnitude.[13]

Furthermore, real-time PCR assays have been demonstrated to exhibit very high precision with coefficients of variance usually in the range of a few percents. When this HCV-specific single-tube two-round real-time RT-PCR assay was analyzed for its precision, coefficients of variance of about 10% were obtained.[12] Nevertheless, the developed assay sufficed for the purpose of sensitive detection of HCV RNA in clinical samples, which was not achieved with one-round real-time RT-PCR.[14] As the reaction tubes remained closed during the whole procedure including RT, first- and second-round PCR, and product detection by melting curve analysis, carryover contaminations into samples analyzed in parallel were precluded.

Getting Started With Dumbbells

Getting Started With Dumbbells

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.

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