Site-directed Mutagenesis를 이용한 GFP 돌연변이 도입 및 DNA 염기서열 분석

본 내용은 " [A+] 2024 서강대 현대생물학실험2 4차 (site directed mutagenesis) " 의 원문 자료에서 일부 인용된 것입니다. 2025.07.01

문서 내 토픽

• 1. Site-directed Mutagenesis

  Site-directed mutagenesis는 DNA 서열의 특정 부분에 point mutation, deletion, insertion 등의 돌연변이를 도입하는 방법이다. 이 실험에서는 mutation을 포함하는 DNA primer를 이용하여 pGFPuv에 Y66H와 Y145F mutation을 도입했다. PCR을 통해 open circular DNA를 생성한 후 DpnI 효소로 메틸화된 template DNA를 선택적으로 제거하여 돌연변이를 도입한다. Pfu DNA polymerase는 3' to 5' exonuclease 활성으로 높은 정확도를 제공한다.

• 2. GFP와 Chromophore 구조

  GFP(green fluorescent protein)는 해파리에서 분리된 형광 단백질로 395nm에서 excitation, 509nm에서 emission한다. 11개의 β-strand로 이루어진 β barrel 구조의 중앙에 chromophore가 존재하며, 65번 Ser-66번 Tyr-67번 Gly 서열로 형성된다. 145번 Tyr은 chromophore와 상호작용한다. Y66H mutation은 파란색 형광을, Y66H/Y145F double mutation은 더 진한 파란색 형광을 발현한다.

• 3. DNA Sequencing 기술

  DNA sequencing은 DNA의 염기 순서를 결정하는 생화학적 방법이다. Sanger sequencing은 ddNTP에 의해 DNA 중합반응이 종료되는 원리를 이용하며, chain termination method와 dye-terminator method로 나뉜다. Next generation sequencing은 한 번에 많은 DNA 절편을 sequencing하며 Illumina, Ion torrent, Roche 454, SOLiD 등의 방법이 있다. 이 실험에서는 Finch TV로 sequencing 결과를 확인하고 BLAST로 유전자를 분석했다.

• 4. Transformation과 Selection

  Site-directed mutagenesis로 도입한 mutation은 BL21 competent cell에 transformation하여 확인한다. BL21은 recombinant protein 생산에 주로 사용되며 high-level protein expression이 특징이다. pGFPuv는 ampicillin 저항성 유전자를 포함하여 selection marker로 사용된다. Heat shock 후 회복 시간과 plasmid DNA의 비율 조정으로 transformation 효율을 향상시킬 수 있다.

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• 1. Site-directed Mutagenesis

  Site-directed mutagenesis is a powerful molecular biology technique that enables precise modification of specific DNA sequences. This method is invaluable for studying protein function, improving enzyme properties, and creating disease models. The technique has evolved significantly from its inception, with modern approaches like QuikChange and CRISPR-based methods offering improved efficiency and accuracy. The ability to introduce targeted mutations allows researchers to understand structure-function relationships at the molecular level. However, the technique requires careful primer design and optimization to minimize off-target mutations. Despite these challenges, site-directed mutagenesis remains essential for synthetic biology, protein engineering, and fundamental research applications.

• 2. GFP와 Chromophore 구조

  Green Fluorescent Protein and its chromophore represent a remarkable discovery that revolutionized biological imaging. The chromophore, formed through autocatalytic cyclization of three amino acids (Ser65-Tyr66-Gly67), demonstrates nature's elegant solution for fluorescence generation without requiring external cofactors. Understanding GFP's chromophore structure has enabled development of numerous fluorescent variants with different spectral properties. The structural insights have facilitated protein engineering to create improved versions with enhanced brightness and photostability. GFP's discovery earned the Nobel Prize, highlighting its significance in modern biology. The chromophore's unique properties continue to inspire development of new biosensors and imaging tools, making it fundamental to contemporary cell biology and biomedical research.

• 3. DNA Sequencing 기술

  DNA sequencing technology has undergone revolutionary transformations, from Sanger sequencing to next-generation sequencing platforms. Modern sequencing enables rapid, cost-effective determination of genetic information, democratizing genomic research. High-throughput sequencing has accelerated discoveries in genomics, transcriptomics, and metagenomics. The decreasing costs have made whole-genome sequencing accessible for clinical diagnostics and personalized medicine. However, challenges remain including handling massive data volumes, ensuring accuracy in repetitive regions, and interpreting variants of uncertain significance. Long-read sequencing technologies are addressing some limitations of short-read methods. The continuous advancement of sequencing technology promises deeper insights into genetic diversity, disease mechanisms, and evolutionary biology, making it indispensable for modern biological research.

• 4. Transformation과 Selection

  Transformation and selection are fundamental techniques for introducing foreign DNA into cells and identifying successfully transformed organisms. These complementary processes are essential for genetic engineering, creating transgenic organisms, and producing recombinant proteins. Transformation methods vary depending on cell type, including chemical competence, electroporation, and microinjection, each with specific advantages. Selection strategies using antibiotic resistance markers or fluorescent proteins enable efficient identification of transformed cells. The combination of these techniques has enabled creation of genetically modified crops, production of therapeutic proteins, and development of disease models. Modern approaches increasingly employ selectable markers that minimize environmental concerns. Despite their widespread use, optimization remains necessary for different organisms and applications, making transformation and selection critical skills in molecular biology and biotechnology.