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Codes sequencher letters6/17/2023 ![]() Quantitative PCR conditions included 40 cycles of 95☌ for 10 seconds, 55☌ for 30 seconds, and 72☌ for 15 seconds. Reverse transcription-quantitative PCR (RT-qPCR) was carried out in a total volume of 10 μL containing 5 μL DyNAmo Color Flash SYBR Green qPCR (Thermo Scientific), 0.5 μM each primer, and 0.1 μg complementary DNA (cDNA). Primers B-A_F (5′-CACCACACCTTCTACAATG-3′) and B-A_R (5′-TAGCACAGCCTGGATAG-3′) were applied to amplify the internal control β-actin gene. The quantitative expression profiles of both wild-type and mutant alleles of DOCK9, IPO5, and STK24 were assessed using the specific primers listed in the Table. Plasmid DNA for carrying out the transfections was extracted from overnight cultures with a GenElute HP Plasmid Maxiprep Kit (Sigma-Aldrich). The sequence data were analyzed using Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA). The integrity of the clones was confirmed by Sanger sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA, USA). The colonies containing the recombinant vectors were selected using colony PCR. After purification with a GenElute PCR Clean-Up Kit (Sigma-Aldrich), the PCR fragments were ligated into the pTargeT vector using the pTargeT Mammalian Expression Vector System (Promega, Mannheim, Germany) and transformed into JM109 competent cells according to the manufacturer's protocol. Detailed protocol and thermal conditions are available upon request. Polymerase chain reaction analyses were performed using Taq DNA polymerase in a final volume of 25 μL (Thermo Scientific, San Jose, CA, USA). Our results indicate that further elucidation of the causes of KTCN is needed.įor the in vitro splicing experiments, genomic DNA samples from an affected individual (KTCN-014-16) and from an unaffected individual not carrying mutations (KTCN-014-01) were amplified using primers specific for the regions of interest in DOCK9, IPO5, and STK24, as shown in the Table. However, because the mutation effect was observed in vitro, a definitive relationship between DOCK9 and KTCN phenotype could not be established. ![]() The exon skipping causes a premature stop codon, disrupting the functional domains of DOCK9 protein, which may alter the biological role of DOCK9 as a Cdc42 activator.īased on in vitro results, we demonstrated that c.2262A>C substitution in DOCK9, previously identified in KTCN-affected members of an Ecuadorian family, leads to a splicing aberration. In vitro splicing analysis revealed that only c.2262A>C in exon 20 of DOCK9 led to aberrant splicing, resulting in the changed ratio between two protein isoforms: a normal transcript and a transcript with exon skipping. ![]() After transfecting HeLa cells with each construct, total RNA samples were extracted, reverse transcribed, and amplified using specific primers. We generated expression constructs using patient DNA as a template corresponding to the wild-type and mutant alleles of DOCK9, IPO5, and STK24. As the pathogenic consequences of these variants were not obvious, we performed in vitro splicing analyses to determine their functional significance. Previously, we identified heterozygous single base-pair substitutions in DOCK9, IPO5, and STK24, showing concurrent 100% segregation with the affected phenotype in an Ecuadorian family. Keratoconus (KTCN) is a degenerative disorder of the eye that is characterized by a conical shape and thinning of the cornea, resulting in impaired visual function. ![]()
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