Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.

An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 µM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR.

Two chloroplast DNA inversions originated simultaneously during the early evolution of the sunflower family (Asteraceae).

The chloroplast DNA (cpDNA) inversion in the Asteraceae has been cited as a classic example of using genomic rearrangements for defining major lineages of plants. We further characterize cpDNA inversions in the Asteraceae using extensive sequence comparisons among 56 species, including representatives of all major clades of the family and related families. We determine the boundaries of the 22-kb (now known as 22.8 kb) inversion that defines a major split within the Asteraceae, and in the process, we characterize the second and a new, smaller 3.3-kb inversion that occurs at one end of the larger inversion. One end point of the smaller inversion is upstream of the trnE-UUC gene, and the other end point is located between the trnC-GCA and rpoB genes. Although a diverse sampling of Asteraceae experienced substantial length variation and base substitution during the long evolutionary history subsequent to the inversion events, the precise locations of the inversion end points are identified using comparative sequence alignments in the inversion regions. The phylogenetic distribution of two inversions is identical among the members of Asteraceae, suggesting that the inversion events likely occurred simultaneously or within a short time period shortly after the origin of the family. Estimates of divergence times based on ndhF and rbcL sequences suggest that two inversions originated during the late Eocene (38-42 MYA). The divergence time estimates also suggest that the Asteraceae originated in the mid Eocene (42-47 MYA).