Advantages and Disadvantages of PTT

Despite the fact that chain-truncating mutations can be detected by conventional scanning methods such as DNA sequencing or single-strand conformation polymorphism analysis (SSCP), PTT remains the method of choice for many laboratories. This is primarily due to its ability to accurately scan a large sequence (as large as 3 kb) in a single reaction. Once a chain truncation is identified and its position on a gel is known, a much smaller region of DNA can be scanned by sequencing to confirm and characterize the mutation. Although PTT detects only chain truncating mutations, these mutations are invariably associated with disease, especially in the case of tumor suppressor genes because truncated proteins are almost always nonfunctional. In contrast, gene variants that result only in an amino acid substitution are often difficult for the clinician to interpret without additional information.

However, there are a variety of limitations for the PTT approach. First, PTT is usually only appropriate for those genes that contain a high proportion of protein truncating mutations. In addition, it is only effective for large exons as protein fragments encoded by smaller exons are not easily analyzed by SDS-PAGE. This limitation can be overcome by using mRNA as the starting material. However, there are handling and storage concerns with the use of mRNA due to nonsense-mediated mRNA decay[16] and the lack of mRNA expression in accessible tissue (peripheral blood). In addition, PTT cannot detect mutations occurring outside the coding region, such as those that affect control of gene expression.

A number of problems can arise during the PCR amplification step used in PTT. For example, if the mutated allele has a large insertion in the amplicon or if the mutation is a deletion that includes the primer binding site, the mutant allele will not amplify. Also, polymerase error in the first few cycles can lead to an artifact that can be mistaken for an authentic mutation. Artifacts can also be produced from in vitro translation and include false-initiation from internal ATG codons (which is more severe in Escherichia coli-derived reaction mixtures) and proteolytic degradation of the full-length protein by endogenous proteases present in the in vitro synthesis lysate. These and other factors can give rise to background bands in the wild type control samples which can interfere with detection of an overlapping mutant band.

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.

Get My Free Ebook


Post a comment