對 HLA-B*2704 單核苷酸多態性變異基因於聚合酶連鎖反應錯誤複製因素之探討

Investigation of Error Replication Factors in the Polymerase Chain Reaction for HLA-B*2704 Single Nucleotide Polymorphic Variants

劉展銘(CHAN-MING LIU)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程學系/碩士(2024年)

https://doi.org/10.6840/cycu202400474

摘要

僵直性脊椎炎為一種慢性自體免疫疾病，與基因HLA-B27中單核苷酸多態性變異(Single Nucleotide Polymorphism, SNP)相關，其中出現致病率最高之基因為HLA-B*2704。因此診斷僵直性脊椎炎與否的其中一個方法，即為檢測DNA中的HLA-B*2704是否存在SNP，可以大幅降低誤診的機率以及錯誤的用藥。
之前本研究室已發展出使用一種不全互補之引子，成功辨別HLA-B*2704之SNP，本研究則進一步於聚合酶連鎖反應(PCR)中，使用不同序列之正向引子，其序列一樣與模板HLA-B*2704不全互補。實驗集中在探討所設計之引子，是否於聚合酶連鎖反應中擴增出正確長度之產物。若未正確複製，則探討出一般的通則，在什麼情況下會進行錯誤的DNA複製，產生錯誤的感測結果。實驗於電泳膠上發現過於相似的序列會造成錯誤的擴增，但修改後的正向引子可修正錯誤。實驗進一步於正向引子5’端修飾Biotin，反向引子於5’端修飾 Digoxigenin，再對其PCR產物於側向流免疫層析試條上，測試不同的試劑濃度所產生之感測訊號。最終經實驗結果顯示，最佳的試劑濃度為：Lateral flow buffer 500 mL中含10X PBS 50 mL。本研究亦對不同的硝化纖維膜進行比較，經實驗結果發現，不同的硝化纖維膜在相同條件下的相異性非常小，幾乎可忽略不計。

關鍵字

單核?酸多態性變異 ； 側向流試片 ； HLA-B2704

並列摘要

Ankylosing spondylitis is a chronic autoimmune disease associated with single nucleotide polymorphism (SNP) variations in gene HLA-B27. In this gene group, HLA-B*2704 allele exhibits the highest pathogenicity. Consequently, the most effective methodology to diagnose ankylosing spondylitis has to involve the detection of the presence of SNPs in HLA-B*2704. This approach is significantly able to reduce the possibility of misdiagnosis and incorrect medication.
In our previous researches, primers with partially complementary sequences with the PCR template were successfully developed to identify the SNP of HLA-B*2704. However, there was no investigation for the incorrect PCR amplification during the development work. In this study, various forward primers with partially complementary sequences with HLA-B*2704 template were employed to run the polymerase chain reaction(PCR). The focus of the experiment was to determine whether or not the designed primers could amplify PCR products with the correct length. For the case that the correct replication was not achieved, we aimed to find out the general rules why incorrect DNA replicates generate, which further leads to incorrect bio-sensing results on the lateral-flow immunoassay strips. The experiment revealed that highly similar sequences at two ends of the primers cause incorrect PCR amplification. The incorrect amplification was found can be eliminated by removing the sequence with similarity from the forward primers. After the assurance of correct PCR production, the forward primers were further modified with Biotin at the 5' end, and the reverse primers with Digoxigenin, respectively to generate a PCR product with detection labels.
This study also detected the PCR products on lateral-flow immunoassay strips by different ionic concentrations of the running buffer. The results indicated that the optimal buffer ionic concentration was made by adding 50 mL of 10X PBS in 500 mL of lateral flow buffer. Additionally, two different brands of nitrocellulose membrane were compared their performance under identical experimental conditions. They were found negligible differences in conclusion.

並列關鍵字

SNP ； LFIA ； HLA-B2704

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