La visión del PCE ante las últimas penas de muerte

The sequence data results were converted into Chromas Lite and GeneDoc files and the positions of the sequences were identified along the ovine calpain 3 sequence (AF087570). The base sequences of calpain 3 exon-10 PCR amplicons of each genotype were identified, and they were identical to the genotype sequences which had been reported in GenBank as DQ660376~378 (Zhou et al., 2007). The position of obtained sequences was found along ovine calpain 3 (AF087570), which was 1238~1402bp.

Three base substitutions have been identified at position 1263, 1332, and 1344bp within exon- 10 that correlated with three alleles. The sequence of the AA-genotype at these three positions was C/G/C, the BB-genotype was C/T/T and the CC-genotype was T/T/C (Figure 5.6).

Figure 5.6 Calpain 3 exon-10 sequence results.

Sequence results were converted to Chromas Lite to see the sequence determined by the peak of each base (top). Positions of the sequence were identified using GeneDoc (bottom) based on the ovine calpain 3 sequence (AF087570). Arrows indicate base differences among three alleles. A=adenine, T=thymine, G=guanine and C=cytosine.

There were no other base substitutions between AA and BB-genotypes in the complete cDNA sequences. However, base differences between AA and CC-genotypes were identified at six positions including the two positions within exon-10, at 1263bp and 1332bp, and four others at 1491bp, 2127bp, 2331bp and 2433bp (Table 5.5).

Table 5.5 Base differences among the three genotypes.

The positions of base differences between the AA, BB and CC-genotypes were identified. Base differences within exon-10 are indicated (Ex10). Positions are based on the ovine calpain 3 sequence (AF087570). n=number of sequence results/number of samples prepared for sequencing. For example, T n=4/5 means 4 results showed T out of 5 sample prepared for sequencing. A=adenine, T=thymine, G=guanine and C=cytosine.

Because of the low frequency of the C-allele, AC heterozygote samples were also sequenced as an alternative way to identify the C haplotype gene sequence. The AC-genotype should show a mixture of A and C sequences. Once the AA-genotype sequence was determined, sites in AC that had double base pairs could also be used to identify the C sequence as the

alternative to the A sequence at these positions. To confirm this approach, exon-10 PCR products of AA and AC-genotypes were sequenced. The AA-genotype sequence was C/G/C and CC-genotype had T/T/C at 1263, 1332, and 1344bp, respectively. The AC-genotype showed Y/K/C (code Y means C or T and code K means T or G). When the AC-genotype was sequenced, corresponding results were shown at other positions of base substitution between AA and CC-genotype, except 2433bp (Table 5.6). At 1491bp, AA-genotype showed C, and CC-genotype showed T, while AC-genotype showed Y which means C or T. At 2127bp and 2331bp, AA-genotype showed G and A, and CC-genotype showed A and G, while AC- genotype showed R which means A or G.

Table 5.6 Base differences between A and C-allele.

The positions of base differences among the AA, AC and CC-genotypes were identified. Base differences within exon-10 are indicated (Ex10). Positions are based on ovine calpain 3 sequence (AF087570). n=number of sequence results/number of samples prepared for sequencing. A=adenine, T=thymine, G=guanine, C=cytosine, Y=C or T, K=T or G, and R=A or G.

None of base substitutions between AA and CC code for amino acid substitutions, so the amino acid sequences resulting from the AA, BB and CC-genotypes are identical. However there were other consistent differences from the registered calpain 3 mRNA

sequence (AF087570). There were 15 base substitutions found between genotype samples and the registered ovine sequence, at 663, 666, 681, 687, 707, 723, 725, 746, 748, 1761, 1862, 1963, 1966, 1979, and 1993bp and eight of these substitutions coded for amino acid

substitutions. These differences do not include the positions of allele variations in exon-10. The substitutions at 707bp and 725bp caused substitutions of glycines for alanines. At 1966bp and 1993bp, there were substitutions of lysines for glutamic acids. Amino acid substitutions of glutamic acid for glycine, leucine for valine, glycine for valine, and isoleucine for

Table 5.7 Base differences between genotype samples and the registered ovine calpain 3 mRNA sequence (AF087570).

Obtained sequences were compared with the registered ovine calpain 3 sequence (AF087570). The positions of base differences causing amino acid differences are indicated with the names of the amino acids. A=adenine, T=thymine, G=guanine and C=cytosine. OV=ovine calpain 3 (AF087570). Geno=homozygote samples has been tested in this research.

5.4

Discussion

5.4.1 Amplification of calpain 3 exon-10 from genomic DNA

Calpain 3 exon-10 sequences were amplified for SSCP analyses to identify genotypes using genomic DNA extracted from dried blood spots on filter papers as templates. The polymerase chain reaction (PCR) is a very commonly used technique to amplify target gene sequences (Mullis & Faloona, 1987). It involves three steps; denaturation of template DNA, primer annealing and extension to reconstruct the target sequence.

Traditionally, DNA has been extracted from white blood cells using a phenol-chloroform method (Gross-Bellard, Oudet, & Chambon, 1973) or a “salting-out” rapid purification procedure (Miller, Dykes, & Polesky, 1988). However, these methods are time consuming and expensive, designed to produce far more purified DNA than is necessary for rapid PCR diagnosis. The use of dried blood spots on filter paper is a better alternative for PCR-based

genetic screening, because of the convenience of handling and storage of large number of samples (da Silva, Gontijo, Pacheco Rda, & Brazil, 2004; Majumdar, Rehana, Jumah, & Fetaini, 2005; Rock et al., 2005). There are several protocols for extracting DNA from dried blood spots on filter paper, such as the methanol method (Caggana, Conroy, & Pass, 1998), the protease K method (Panteleeff et al., 1999), the Chelex-100 method (Polski, Kimzey, Percival, & Grosso, 1998), and the TE method (Bereczky, Martensson, Gil, & Farnert, 2005). However these methods require multiple washing steps which increase the risk of cross contamination during multistep pipetting.

In these experiments, genomic DNA was extracted from blood spots dried onto filter paper, using sodium hydroxide (Zhou et al., 2006). This method requires only one incubation step followed by a single wash to yield genomic DNA sufficiently pure to be a template for PCR amplification (Zhou et al., 2006).

Calpain 3 exon-10 was amplified using genomic DNA extracted from dried blood samples on filter paper within a week, or from blood samples on FTA cards within a year. The expected size of the calpain 3 exon-10 PCR amplicons was 164bp. The PCR amplicons showed one clear band at around 170bp on the agarose gel (Figure 5.2), indicating that the calpain 3 exon- 10 sequence was successfully amplified, the genomic DNA extracted by the sodium

hydroxide method was reliable to be used as template, and the method could be applied to the fresh or old blood samples on filter paper or FTA cards.