AGRONOMIJAS VĒSTIS - Latvijas Lauksaimniecības universitāte

AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.10, LLU, 2008IntroductionThe Severe combined immunodeficiency disease (SCID) of horses is an autosomal,recessive hereditary disease occurring among Arabian horses or part-bred Arabian horses. SCIDaffectedfoals always die within the first six months of life.The first identification of SCID was made in the 1973 (McGuire & Poppie et al., 1973).The mode of inheritance of SCID has been studied using test mating which showed that SCID iscaused by a simple recessive mutation (Perryman et al., 1978; Perryman & Torbeck et al., 1980).The underlying defect in Arabian horses with SCID is an inability to carry out V(D)Jrecombination (Wiler et al., 1995), and they have been found to lack the enzyme DNA-dependedprotein kinase (DNA-PK). Sequence analysis revealed a 5 base pair deletion within the catalyticsubunit of DNA-PK. This deletion causes a frameshift mutation at codon 3155 of the transcript,resulting in a 967 amino acid deletion, and causing a functionally inactive enzyme (Shin et al.,1997; Perryman, 2004). This gene is localized in 9-Th chromosome (Eca 9p12) (Wiler et al., 1995;Bailey et al., 1997).Materials and MethodsDNA extracted from animal blood samples (in EDTA or sodium citrate) or hair bulbs. TheDNA educed from blood using 5% Chelex-100. A few drops of liquid blood or a few fibers of thedried blood irrigated with a normal saline solution. It was extracted during a time period of 1 hourunder 65 ○ С with 200µl 5% Chelex-100. The time of the incubation period could have been longer.The solution of the DNS was possible use for PCR analyses after 5 minutes of incubation under100 ○ С and centrifugation (Lewin et al, 1992).The bulb was extracted from the hair by two different methods. The first method -extraction using 5% Chelex-100 - resulted in low DNK concentration in the final solution.Therefore, another method has been chosen - extraction using commercial whale GenepakMoscow. 4-5 bulbs were incubated for 5 minutes at 65 ○ C after adding 300 µl of Lysis reagent withmixing every 30 seconds. After centrifugal separation 20 µl of NucleoS, vortex 5 min was added tothe solution. The sediment was washed out 3 times using a saline solution and then dried out. Thecarier has to be incubated with 100 µl ExtraGene at 65 ○ C for 5 minutes. The derived solution maybe used for PCR analysis.The polymerase chain reaction was performed in a GeneAmp PCR System 2400 (PEApplied Biosystem). We used 2 couple of praimers.One set of allele-specific praimers (SCID1) was designed to amplify only DNA containingthe 5-basepair deletion and another set designed to amplify only normal allele (SCID2). Theforward primer (SCID3) was identical for both sets (Table 1). The reaction tests were carried out in10µl final volume containing 9,5 µl PCR mix (final concentration: 10 mM Tris-HCl, pH 8,3; 50mM KCl; 1,5 mM MgCl 2 ; 125 µM dNTP; 0,5 µM of each praimer; 0,35 units of Taq polymerase)and 0,5 µl of DNA samples.Table 1. DNA praimers (Bernoco et al., 1998)SCID1 praimer 3`-TAGTTTAAGGGGAATTCTCTGAA-5`SCID2 praimer 3`-AGAGTTTAAGGGGAATTCTCTG-5`SCID3 praimer 3`-TGTTGCAAAAGGAGACAGAAT-5`The following cycle regime was used: 94 ○ C for 10 minutes; 94 ○ C for 30 seconds denaturation,61 ○ C for 30 seconds annealing, 75 ○ C for 30 seconds elongation for 33 cycles.The DNA primers, SCID4 and SCID5, (Table 2) which flank the gene region affected bythe mutation are used in a polymerase chain reaction (PCR) to amplify the affected region from theDNA sample. Composition of the miscellanies for the reaction mix was similar as indicated above.Table 2. DNA praimers (Swinburne et al., 1999)SCID4(fr) praimer 5`-AAGTTGGTCTTGTCATTGAGC-3`SCID5 (rev) praimer 5`-TTTGTGATGATGTCATCCCAG-3259