The PCR mixture (50 μL) for amplifying the full-length mutant gene contained 40 ng of each megaprimer, 10 μL of 5× GC buffer, 3% DMSO, 0.4 mM dNTPs, and 1 unit of Phusion DNA polymerase.
Bhuvanagiri et al (2014) have identified a means to allow mutant genes containing aberrant stop codons to become useful.
Moreover, approximately one-third of inherited genetic disorders and many forms of cancers are associated with mutant genes containing PTCs [ 23- 26].
The human γD-crystallin (CRYGD) and mutant genes containing N-terminal hexa-His tags were inserted into the pQE.1 vector (Qiagen), and protein was produced in transformed Escherichia coli M15 cells, essentially as described previously (26).
Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer.
To meet these requirements, we have generated transgenic mice harboring a single copy of the full-length human mutant DRPLA gene containing 76 CAG repeats (13).
The third mutant envB gene contained both the A32V and Δ152 164 mutations (Fig. 2A).
Soybean codon optimized mutant SEB genes containing sequences encoding the native SEB N-terminal signal peptide sequence and the native soybean glycinin N-terminal signal peptide sequence were synthesized from GeneArt (Life Technologies Carlsbad, CA) and DNA 2.0 (Menlo Park, CA), respectively.
Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.
In patients with myotonic dystrophy type 1 (DM1), an adult-onset form of muscular dystrophy caused by the expression of mutant transcripts from the DMPK gene containing expanded CUG repeats [11] [13], CELF1 is aberrantly up-regulated in the heart and skeletal muscles [10], [14].
A control reporter gene containing five mutant c-Myb binding sites (EM5) was much less active in these cells, and its activity was unaffected by the different forms of v-ErbA.
