Material and Methods

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Genomic DNA Sources of Human AMELX/AMELY and PCR

Amplification

Human genomic DNAs used for AMELX were taken from 32 unrelated individuals (19 males and 13 females) in a worldwide sample in Coriell Cell Repositories (http://locus.umdnj.edu/ccr/): 12 Africans (6 Biaka Pigmies, 4 Mbuti Pygmies, and 2 Blacks), 8 Eastern Europeans (4 Adygeis and 4 Russians), 4 Middle Easterns (Druze), 4 Asians (Ami), and 4 Amerinds (2 Mayan and 2 Kari-tiana). The repository numbers are NA10469-10473, 1049210496, 10968, 10969, 10975, 10976, 11521-11524, 13607-13610, 13617-13620, 13820, 13838, 13849, 13877, 14537, and 14661. Those DNAs were used as templates for PCR amplification of AMELX (6513 bp). The PCR primers were designed within the promoter region and at the end of exon 6. The upper and lower primer sequences are 5'-GCTGCTGAGAACATGGAA-3' and 5'CAC-TTCCTCCCGCTTGGTCTTGTC-3', respectively. Similarly, 18 unrelated males were used for AMELY: 5 Africans (1 Biaka Pigmy,

3 Mbuti Pygmies, and 1 Black), 4 Eastern Europeans (2 Adygeis and 2 Russians), 1 Middle Eastern (Druze), 4 Asians (Ami), and

4 Amerinds (2 Mayan and 2 Karitiana). The repository numbers are NA 10470, 10492, 10494, 10495, 10968, 10969, 10975, 10976, 11522, 13607-13610, 13619, 13620, 13820, 13838, and 14537. The PCR primers of genomic AMELY sequences (6627 bp) were designed within the promoter region and at the end of exon 5. Unfortunately, exon 6 had to be excluded for technical reasons. The upper and lower primer sequences are 5'-AAGTGCCATGTGGT-GAATTAGG-3' and 5'-GTACCAGAGCATGATAAGA-3 ', respectively.

Each PCR reaction mixture was 50 ^l in volume and contained 15 ng of genomic DNA, Platinum PCR SuperMix High

Fig. 1. The presence or absence of the AMEL gene within the Rho GTPase-acti-vating protein 6 gene (ARHGAP6) in humans, opossums, chicken, Xenopus tropicalis and Tetraodon nigroviridis. In chicken, it appears that exon 1 and the following intron of ARHGAP6 are deleted together with AMEL.

Fig. 1. The presence or absence of the AMEL gene within the Rho GTPase-acti-vating protein 6 gene (ARHGAP6) in humans, opossums, chicken, Xenopus tropicalis and Tetraodon nigroviridis. In chicken, it appears that exon 1 and the following intron of ARHGAP6 are deleted together with AMEL.

Fidelity (Invitrogen, San Diego, Calif., USA), and 0.2 ^M of each primer. The PCR condition was as follows: denaturation at 94°C for 2 min; 34 cycles at 94°C for 15 s, 59°C for 30 s, and 68°C for 5 min. The PCR products were purified using ExoSAP-IT (United States Biochemical, Cleveland, Ohio, USA) and sequenced directly. In the case of heterozygous AMELX, PCR products were cloned into pCR-XL-TOPO with TOPO XL PCR Cloning Kit (Invitrogen). Sequencing reactions were performed with BigDye® Terminator v1.1 and v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, Calif., USA) and analyzed on ABI PRISM 377, 3100, and 3730 DNA sequencer (Applied Biosystems). To avoid sequencing errors, PCR products or plasmid DNAs were read twice in both directions. Sequences were also confirmed by independent PCRs. These fragmental sequences were assembled by DNA-SIS (Hitachi, Yokohama, Japan).

Data Analyses

All DNA sequence alignments were made by Clustal X [Thompson et al., 1997] and then manually checked. For phylo-genetic analyses, the neighbor-joining method [Saitou and Nei, 1987] was used. To evaluate functional importance of AMELX and AMELY, the genomic sequences of primate and non-primate mammalian amelogenin genes were determined [Iwase et al., 2003] and homologous sequences available in the National Center for Biotechnology Information database were retrieved. Unfortunately, most of mammalian AMELX sequences used in Delgado et al. [2005] were unavailable in the DNA sequence database, so that only 20 sequences were used. The ratio (f) of the non-synonymous to synonymous substitutions per site was computed along individual branches in the reconstructed neighbor-joining tree by the least square method [Zhang et al., 1998]. For the human population data, DnaSP v.4.10 [Rozas et al., 2003] was used to compute some summary statistics concerning polymorphism:

the average pairwise nucleotide differences ^ [Nei and Li, 1979], 0 [Watterson, 1975], the normalized ■m - 0 that is defined as D [Tajima, 1989], and linkage disequilibrium that is defined as r2 [Hill and Robertson, 1968].

The HapMap data based on 90 Yorubas (30 parent-offspring trios) in Ibadan and Nigeria (www.hapmap.org) were retrieved. A total of 2,851 single nucleotide polymorphism (SNP) genotypes in X chromosome region were downloaded from the February 2006 public release of the HapMap data. After excluding SNPs with minor variants and missing genotypes, the remaining 1,036 SNPs were used for linkage disequilibrium (LD) map construction by the method of Haploview 3.32 [Barrett et al., 2005].

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