Site-directed mutagenesis & DNA sequencing

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• 1. Site-directed mutagenesis

  Site-directed mutagenesis는 원하는 돌연변이를 포함하는 짧은 DNA primers를 사용하여 DNA 염기서열 특정 위치의 mutation을 일으켜 돌연변이를 얻는 방법입니다. 이 방법을 이용하여 GFP의 chromophore 아미노산 서열을 변경하여 다양한 색상의 형광 단백질을 만들 수 있습니다.

• 2. DNA sequencing

  DNA sequencing은 DNA를 구성하는 염기 순서를 결정하는 작업입니다. Sanger sequencing 방법은 dNTP와 ddNTP의 구조 차이를 이용하여 sequencing을 진행하며, 최근에는 NGS 기술로 대량의 유전체를 동시에 sequencing할 수 있게 되었습니다. 이번 실험에서는 Sanger sequencing 방법을 이용하여 gene X의 DNA 서열을 분석하였습니다.

• 3. GFP 돌연변이체

  GFPuv의 chromophore 아미노산 서열인 65Ser-66Tyr-67Gly에서 66번의 Tyr이 His로 돌연변이된 Y66H mutated GFPuv는 파란색을 띄게 되며, Y66H mutated GFPuv에서 chromophore와 상호작용하는 아미노산인 145번의 Tyr이 Phe로 돌연변이가 일어난 Y66H/Y145F mutated GFPuv는 짙은 파란색을 띄게 됩니다.

• 4. Gene X 분석

  이번 실험에서 다루었던 Gene X는 720개의 염기서열을 가지며, 239개의 아미노산으로 이루어진 GFP superfamily의 GFP(Green fluorescent protein)입니다. Gene X의 아미노산 서열은 SGFP2(synthetic construct fluorescent protein)와 99.17% 유사하며, 65번 아미노산 Serine이 Alanine으로 변이되어 있습니다.

• 5. Pfu DNA polymerase

  Pfu DNA polymerase는 Pyrococcus furiosus에서 유래되었고, 3' to 5' exonuclease activity를 갖고 있어 DNA 합성 중 오류를 수정할 수 있습니다. 따라서 Taq DNA polymerase보다 PCR 진행 시 오류가 일어날 가능성이 낮아 Site-directed mutagenesis에 적합합니다.

• 6. Shine-Dalgarno sequence

  Prokaryote의 translation은 개시코돈에서 5' 방향으로 앞쪽에 Shine-Dalgarno sequence인 5'-AGGAGGU-3'가 존재하여 16S rRNA가 base-pairing에 의해 mRNA를 aligns합니다. 이번 실험에서 Gene X의 첫번째 ATG 앞에 ribosome binding site로 5'-AAGGAG-3'이 존재하였습니다.

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• 1. Site-directed mutagenesis

  Site-directed mutagenesis is a powerful molecular biology technique that allows researchers to introduce specific, targeted changes in the DNA sequence of a gene or plasmid. This method is widely used in various fields, including protein engineering, functional genomics, and the study of structure-function relationships. By precisely modifying the genetic material, scientists can investigate the effects of particular amino acid substitutions, insertions, or deletions on the structure, function, and expression of a protein of interest. Site-directed mutagenesis enables the creation of novel enzymes, the study of disease-associated mutations, and the optimization of protein properties for various applications, such as biocatalysis, therapeutics, and biotechnology. The ability to control the genetic changes with high specificity is a key advantage of this technique, allowing researchers to gain valuable insights into the underlying mechanisms of biological processes and to develop innovative solutions to complex problems.

• 2. DNA sequencing

  DNA sequencing is a fundamental technique in molecular biology and genetics that has revolutionized our understanding of the genetic makeup of living organisms. This powerful tool enables the determination of the precise order of nucleotides (adenine, guanine, cytosine, and thymine) within a DNA molecule, providing invaluable information about the structure and function of genes, genomes, and genetic variations. The development of various DNA sequencing methods, such as Sanger sequencing, next-generation sequencing, and single-molecule sequencing, has significantly improved the speed, accuracy, and cost-effectiveness of DNA analysis. The ability to rapidly and accurately sequence DNA has enabled groundbreaking discoveries in fields like genomics, evolutionary biology, personalized medicine, and forensics. DNA sequencing has become an indispensable tool for researchers, clinicians, and scientists, allowing them to decipher the genetic code, identify genetic markers, and unravel the complexities of living organisms at an unprecedented scale. As the technology continues to advance, the applications of DNA sequencing will undoubtedly expand, leading to further advancements in our understanding of life and the development of innovative solutions to complex biological problems.

• 3. Gene X analysis

  The analysis of a specific gene, referred to as Gene X, is a crucial step in understanding its function, regulation, and potential implications in biological processes or disease states. Comprehensive gene analysis typically involves a multifaceted approach, including sequence analysis, expression profiling, functional studies, and the investigation of genetic variations. Sequence analysis of Gene X can provide insights into its structure, potential coding regions, regulatory elements, and evolutionary conservation, which can inform hypotheses about its biological role. Expression profiling, using techniques like RT-qPCR or RNA-sequencing, can reveal the spatiotemporal patterns of Gene X expression, shedding light on its developmental or tissue-specific functions. Functional studies, such as gene knockdown or overexpression experiments, can elucidate the phenotypic consequences of modulating the expression or activity of Gene X, thereby elucidating its involvement in cellular pathways or physiological processes. Additionally, the analysis of genetic variations, including single nucleotide polymorphisms (SNPs) or mutations, can uncover associations between Gene X and disease susceptibility or drug response, paving the way for personalized medicine applications. The integration of these diverse analytical approaches can lead to a deeper understanding of Gene X, its biological significance, and its potential as a therapeutic target or biomarker, ultimately contributing to advancements in various fields of life sciences.

• 4. Pfu DNA polymerase

  Pfu DNA polymerase, also known as Pyrococcus furiosus DNA polymerase, is a highly thermostable and accurate enzyme widely used in molecular biology and biotechnology. Derived from the hyperthermophilic archaeon Pyrococcus furiosus, Pfu DNA polymerase is renowned for its exceptional fidelity, with an error rate significantly lower than that of other commonly used DNA polymerases, such as Taq polymerase. This high-fidelity property makes Pfu DNA polymerase an indispensable tool for applications that require precise DNA amplification, such as site-directed mutagenesis, long-range PCR, and the generation of DNA libraries for sequencing. The enzyme's remarkable thermostability, which allows it to function optimally at high temperatures, is particularly advantageous in PCR-based techniques, as it enables efficient DNA synthesis and minimizes the risk of non-specific amplification or primer-dimer formation. Furthermore, Pfu DNA polymerase possesses 3' to 5' exonuclease activity, which confers proofreading capabilities and further enhances the accuracy of DNA replication. The versatility and reliability of Pfu DNA polymerase have made it a staple in molecular biology laboratories, contributing to the advancement of various fields, including genetics, genomics, and biotechnology, by enabling the precise manipulation and analysis of genetic material.

• 5. Shine-Dalgarno sequence

  The Shine-Dalgarno (SD) sequence is a crucial regulatory element in prokaryotic gene expression, playing a vital role in the initiation of translation. Named after the scientists who first described it, the SD sequence is a short, purine-rich nucleotide sequence located upstream of the start codon (AUG) in bacterial and archaeal mRNA. This sequence serves as a binding site for the 16S rRNA component of the small ribosomal subunit, facilitating the recruitment and positioning of the ribosome at the correct translation initiation site. The complementarity between the SD sequence and the anti-Shine-Dalgarno (aSD) sequence in the 16S rRNA helps to ensure that the ribosome recognizes the appropriate start codon and initiates protein synthesis efficiently. The strength and positioning of the SD sequence relative to the start codon can significantly influence the translation efficiency of a gene, as it affects the binding affinity and positioning of the ribosome. Understanding the Shine-Dalgarno sequence and its role in translation initiation has been instrumental in the design and optimization of heterologous gene expression systems in prokaryotes, enabling the production of recombinant proteins and the development of synthetic biology applications. The study of the Shine-Dalgarno sequence continues to provide valuable insights into the fundamental mechanisms of gene expression and translation in prokaryotic organisms.