Association of TLR4-T399I Polymorphism with Chronic Obstructive Pulmonary Disease in Smokers
(a) Representative digestions showing the TLR2-R753Q (A), TLR4-D299G (B), and TLR4-T399I (C) polymorphisms. M: 200 bp ladder molecular weight marker. Lanes 1–8: Patients with COPD; lane 9: negative PCR control. Samples without any polymorphism display undigested PCR products, 430 bp for the TLR2-R753Q, 249 bp for the TLR4-D299G, and 407 bp for the TLR4-T399I. Samples 5 and 7 are heterozygotes for the TLR2-R753Q polymorphism, whereas the TLR2-753Q allele contains a Sfc I restriction site resulting in 307 bp and 123 bp fragments. Samples 2 and 6 are double heterozygotes for both TLR4 polymorphisms. In particular, the TLR4-299G allele contains an NcoI restriction site resulting in 218 bp and 31 bp fragments, while the TLR4-399I allele contains a HinfI restriction site resulting in 375 bp and 28 bp fragments. The digestion products were analyzed on 2% of TBE agarose gels and the 28 bp and 31 bp fragments are not visible on agarose gels. (b) Representative sequencing analysis shows the presence of TLR2-R753Q, TLR4-D299G, and TLR4-T399I polymorphisms.
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