Several factors are important when designing PCR primer pairs for PTT. Proper design of the forward primer is the most critical factor. This primer contains four specific regions that are essential for efficient in vitro translation of the PCR amplicon. The 5'-primer must have a promoter sequence at the 5'-end (generally for T7 polymerase) followed by a 5-7-bp spacer sequence, a eukaryotic translation initiation sequence (Kozak sequence) including the ATG start codon, and at the 3'-end, a region of the target gene sequence (~ 17-24 bp) in-frame with the ATG codon. In addition, for ELISA-PTT, the 5'-primer also contains sequences coding for the N-terminal detection tag (epitope) and/or binding tags. The 3'-primer must be complementary to the 3'-end of target sequence and for ELISA-PTT must also contain a sequence encoding the C-terminal detection tag. To ensure that truncation mutations near the beginning or at the end of a fragment (i.e., at the 5'- or 3'-end) are not missed, flanking segments for a large region of coding sequence should have an overlap of 350-500 bp. When RNA-based PTT is used, primers from overlapping segments should be located in different exons to reduce the possibility that a mutant allele having a deletion or splicing defect does not amplify with any of the primer sets.
Was this article helpful?
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.