癌症基因突變檢測之引子設計整合性平台建置

Abstract

癌症疾病為全球前幾大死因之一，癌症的產生通常與基因上的突變有相當大的關係，而 癌症相關基因的檢測漸漸成為臨床上檢測癌症的重要指標之一，傳統的癌症相關基因檢 測使用 Sanger 定序，然而傳統一個基因一個檢測的方式在現今臨床的使用上造成時間 以及成本上較大的耗損，因此次世代定序技術(Next-generation sequence)漸漸取代傳統 定序的方式，其快速且平行化測序達到同時大量定序的特性成為臨床上較主流的癌症基 因檢測方式。本研究著重於多重引子(Multiple primer)的設計，允許在同一實驗中進行 多重聚合酶鍊鎖式反應(Multiplex PCR)對目標物同時放大，利用次世代定序技術完整定 序出目標物藉由檢測基因變異情況，本平台允許使用者針對所有有興趣之基因同時進行 而引子的設計。 由於基因所有的外顯子都必須被包含，故基因的所有異構物(Isoform)都必須被考慮，如 果有一個外顯子僅出現在一個異構物(Isoform)中，也必須對其做引子(Primer)之設計， 本研究之多重引子設計平台也提供使用者對於不同的參數做設定，如引子的長度、放大 產物之長度、引子 GC 含量以及引子熔化溫度等，本研究引子設計的 GC 含量為>=0.4 且 <=0.6，而引子熔化溫度介於 42℃到 68℃之間找尋熔化溫度最接近的引子集。候選引子 必須介於此熔化溫度之間並且找出溫度最接近之引子集，為了避免引物二聚體(Primer- dimer)在多重聚合酶鍊鎖式反應(Multiplex PCR)中產生，引物二聚體(Primer-dimer)的 檢查也是必要的。最後從候選的引子集(Candidate primer set)中挑出引子熔化溫度最為 接近之引子作為最後實驗的引子集。 本平台同時設計出有興趣之基因或目標物之引子，供多重聚合酶鍊鎖式反應(Multiplex PCR)使用，並期望達到一個時間以及成本節省的多重引子(Multiple primer)設計平台， 所設計出的引子可用來夾出有興趣之目標序列並進行測序，透過生物資訊的分析來檢驗 基因上之變異，增加臨床上應用之價值。

Cancer is one of the top deaths in worldwide, is typically caused by genomic variants or epigenomic alterations. Cancer-associated mutations have became standard tests for clinical diagnosis, which are identified by an orthologous method such as PCR-based Sanger sequencing. An one-gene-one-test or one-mutation-one-test approach can be challenged in clinical application because of its time- and cost-consuming limitation. The next-generation technology allows high throughput in massively parallel sequencing and an amplicon-base NGS approaches has consider as the best method for clinical oncology. This research is focus on multiplex primer design for amplicon-base target sequencing using NGS technology, providing an integrated platform for primer design according to user selected genes. Each isoform of this gene must be considered in. Making the longest exon to be the represented one. The primers are designed for each exon on this gene, even the exon is only existed in one isoform. For primer design, user is allowed to select the length of amplicons and primers. Primer GC ratio should be >= 0.4 and <= 0.6 with melting temperature is between 42℃ and 68℃. The primers should be able to map to the specific location on genome. Primer dimer is checked to reduce self-hybridize in multiplex approach. After all, the closest melting temperature of primer sets will be selected for amplicon-base target sequencing. We believe that this approach is to find out the primer sets for genes which are interested in. A time- and cost-efficient platform for multiplex primer design use in amplicon-base target sequencing is achieved. These primer sets can be used for identifying genetic mutation in cancer-related genes, increase diagnostic value in clinical. The primer sets can be used in PCR-base specific enrichment method to achieve efficient detection of the somatic mutation. A customize, specific, and cost-efficient multiplex primer design platform is constructed for cancer-related gene mutation identification.