Overview

In many diagnostic applications the analytical sensitivity of PCR has been markedly increased, when two rounds of PCR were performed, one PCR after another. In the first round of PCR sample DNA is amplified below the detection limits. Thereafter, the products of the first round of PCR are further amplified to detectable levels in the second round of PCR. For the second round of PCR usually a second set of primers are used which anneal within the DNA sequence of the first-round products. This second set of primers is then termed nested primers or second-round primers. Thus the application of two-round PCR to diagnostic PCR assays has resulted in improved assay sensitivities. In addition, the use of second-round primers increased the specificity of the PCR analysis, because the second-round primers amplify only if the first-round PCR yielded a specific product.[1] So in virus diagnostic applications consensus or indiscriminate primers have been used in a first-round PCR to amplify target DNA, which in a second-round PCR was further amplified with primers that more specifically annealed to the selected viral target DNA. This has increased the sensitivity of PCR analysis to the extent that, for example, in the diagnosis of HIV infection a single copy of HIV-1 cDNA could be detected.[2,3] Furthermore, the two-round PCR technique has successfully been utilized with the simultaneous amplification of various viral genomes such as from herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus. Furthermore, two-round PCR has been applied to the genotyping and subtyping of viral strains.[4] For PCR diagnosis and monitoring of hematological malignancies such as the chronic myelog-enous leukemia, two-round PCR has successfully been employed for the assessment of the chromosomal translocation t(9;22). This has yielded in high sensitivity of detecting up to one malignant cell within 106 normal leukocytes.[5]

As a disadvantage, two-round PCR has suffered from the necessity to recover the products from the first PCR, in order to introduce them into the second-round PCR. During the recovery of first-round products carryover contaminations into samples assayed in parallel may sometimes occur and thus may cause false positive test results to occur. To avoid this, various techniques have been developed to perform two-round PCR in single closed reaction tubes for the conventional PCR technique.[6-8]

In real-time PCR using LightCycler capillaries, the disadvantage of two-round PCR also relates the necessity to recover first-round PCR products. As LightCycler capillaries are rather inaccessible for pipetting, PCR products are usually retrieved by a brief centrifugation step, which releases the capillaries' contents into small tubes. But this procedure is thought to be particularly prone to product carryover. In order to avoid the centrifugation procedure, a two-round real-time PCR technique has been developed for LightCycler capillaries that remain closed during both rounds of PCR.

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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