Fogorvosi szemle, 2007 (100. évfolyam, 1-6. szám)

2007-10-01 / 5. szám

FOGORVOSI SZEMLE

100. évf. 5. sz. 2007.

269

and appropriate quantity of DNA from the oral mucosal scrapings. In the early phase of our studies the samples were frequently degraded. To prevent DNA degradation we modified our sample collection and storage protocols and after collection samples were immediately put into the freezer and stored at -20 °C until processed.

Samples 1, 2 and 3 on Figure 2 show the electrophoretic picture of damaged DNA. Samples 4, 5 and 6 on Figure 2 demonstrate intact, good quality DNA obtained with the modified isolation protocol. The fragment size of DNA that can be regarded as intact is around 20-30 kilobase (kb). As it can be seen in the

for each polymorphism involving both allele variations and also heterozygous examples. The pictures show all PCR-RFLP types that we optimized so far including the wild type, the rare and the heterozygous alleles.

In Figure 3 inserts A-H show the polymorphisms that can be related to periodontitis. The size of the IL- 1 a -889 product is 99 basepairs (bp) that can be digested by Ncol restriction enzyme with C allele with product size 79 bp, while the T allele is not digested (A). The IL-1 ß -511 product size is 305 bp that can be cleaved by Ddel treatment in the case of C allele yielding 160 and 155 bp secondary products, while the T allele gives 114 and 160 bp products (B). The size

\ Periodontal conditions measured by CPITN in relation to gender (‘pcO.Ol, *p<0.1,3p<0.0001)

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Figure 1. Representative data of periodontal conditions the adult Hungarian population by gender (A.), and by age (B.)

Figure, the degraded DNA consists of much smaller fragments. We isolated nearly 70 samples by now in the periodontitis and hypodontia studies. All of these samples can be used in further experiments since they also showed positive results when the presence of the constitutively expressed ß-actin gene was tested.

In our polymorphism studies previously described experimental conditions were taken as the starting point. During checking the sequence of oligonucleotide primers, several of them were proven to be inaccurate. Therefore, we modified these sequences. To optimize PCR reaction conditions, we also modified these parameters as it can be seen in Table I. In some cases we selected new restriction enzymes to receive consistently reproducible results. This way we developed the optimal conditions for each polymorphism study.

We found both allele variations in all potential SNPs in spite of the small sample number. As examples, Figure 3 shows representative electrophoretic pictures

of the IL-1 ß +3954 product is 194 bp. It can be digested by Taql restriction endonuclease producing 73 and 85 bp with C allele and 178 bp with T allele (C). The IL-6 -174 product size is 198 bp that is split by Hsp92ll enzyme into 45 and 122 bp fragments in the case of C allele, and a 167 bp fragment can be seen with G allele (D). The size of the IL-10 -1082 product is 196 bp. Following Mnll restriction enzyme digestion it gives 58 and 110 bp-s with G allele, while 58 and 138 bp with A allele (E). The TNF-a -308 product size is 107 bp that can be cleaved by Ncol treatment in the case of G allele yielding 20 and 87 bp secondary products, while the A allele is not digested. (F). The size of Toll-like receptor -4 (TLR-4) -299 is 249 bp. It can be digested Ncol restriction endonuclease producing 23 and 223 bp with G allele, while the A allele is not digested (G). The TLR4 -399 product size is 407 bp that is digested by Hinfl enzyme into 29 and 378 bp fragments in the case of G allele, while the C allele is not digested (H). When the TLR-4 polymorphisms were inves-