유전학실험 Gene insertion & deletion

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1. PCR (Polymerase Chain Reaction)

PCR은 DNA를 빠르게 증폭시키는 방법으로, Denaturation, Annealing, Elongation의 3단계로 구성된다. Denaturation에서 DNA는 95℃의 고온에 의해 single strand가 되며, Annealing에서 primer가 결합한다. Elongation에서는 약 75℃에서 DNA polymerase의 작용에 의해 DNA의 길이 신장이 일어난다. PCR의 결과값에 영향을 미치는 요인에는 각 단계의 온도, cycle 횟수, buffer solution, primer, DNA polymerase의 종류 등이 있다.
2. Yeast Transformation

Transformation은 exogenous DNA가 cell 내부로 유입되어 유전적 변화나 유전 가능한 변화를 일으키는 것을 의미한다. 이 과정에서 특정한 gene을 삽입할 수도, 반대로 제거할 수도 있다. 일반적으로 사용되는 transformation 방법에는 lithium, electroporation, glass bead and biolistic (gene gun) method가 있다. DNA가 Yeast의 내부에 들어간 뒤 Yeast의 자체 DNA 복원 기작(HR)에 의해 genomic DNA로 삽입한 DNA가 끼어들어간다.
3. Transformation Efficiency Factors

PEG는 cell wall을 약화시키며 yeast cell의 표면에 DNA가 결합할 수 있게 한다. LiAc는 cell wall과 cell membrane에 구멍을 뚫어 DNA가 잘 들어갈 수 있도록 효율을 올려준다. Salmon sperm DNA는 yeast 내부에 들어가면 DNA repair system을 활성화시켜 transformation 효율을 높인다.
4. Confirmation of Gene Modification

Yeast transformation을 통해 만들어진 modification gene이 실제로 맞는지 확인하기 위해서는 DNA 자체를 확인하는 Sanger sequencing 방법과, 발현된 protein을 확인하는 Western blot 방법이 존재한다.

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1. PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction) is a powerful molecular biology technique that allows for the exponential amplification of specific DNA sequences. It has revolutionized various fields, including genetics, forensics, and diagnostics. The ability to rapidly and accurately replicate DNA fragments has enabled researchers to study and analyze genetic material in unprecedented ways. PCR has become an indispensable tool in fields such as disease diagnosis, genetic profiling, and evolutionary studies. The technique's sensitivity and specificity make it a crucial component in modern molecular biology research and applications. As technology continues to advance, the potential applications of PCR will only continue to grow, further expanding our understanding of the genetic underpinnings of life.
2. Yeast Transformation

Yeast transformation is a fundamental technique in molecular biology and biotechnology, allowing for the introduction of foreign genetic material into yeast cells. This process enables researchers to study gene function, express heterologous proteins, and engineer yeast strains for various applications. Yeast, as a eukaryotic model organism, offers several advantages, including its well-characterized genome, ease of genetic manipulation, and rapid growth. The ability to transform yeast cells has facilitated advancements in areas such as recombinant protein production, metabolic engineering, and the development of yeast-based biofuels and pharmaceuticals. As our understanding of yeast biology and genetics continues to expand, the applications of yeast transformation will likely continue to grow, contributing to advancements in both basic and applied research.
3. Transformation Efficiency Factors

Transformation efficiency is a critical factor in the successful introduction of foreign genetic material into host cells, whether they are bacteria, yeast, or other organisms. Understanding the various factors that influence transformation efficiency is essential for optimizing genetic engineering protocols and achieving desired outcomes. These factors can include the competence of the host cells, the quality and concentration of the DNA being introduced, the method of DNA delivery (e.g., heat shock, electroporation), and the environmental conditions during the transformation process. By carefully controlling and manipulating these factors, researchers can maximize the number of successfully transformed cells, which is crucial for applications such as protein production, gene expression studies, and the development of genetically modified organisms. Continued research into transformation efficiency factors will help streamline and improve the reliability of genetic engineering techniques, further advancing our capabilities in various fields of biotechnology and molecular biology.
4. Confirmation of Gene Modification

Confirming the successful modification of a gene is a critical step in genetic engineering and biotechnology. Accurate verification ensures that the desired genetic changes have been correctly introduced and that the resulting organism or cell line exhibits the intended phenotypic characteristics. Various techniques, such as PCR, DNA sequencing, and reporter gene assays, are commonly employed to confirm gene modification. These methods allow researchers to validate the presence, sequence, and expression of the introduced genetic material. Robust confirmation protocols are essential for ensuring the reliability and reproducibility of genetic engineering experiments, as well as for regulatory compliance in the development of genetically modified organisms or products. As genetic engineering technologies continue to advance, the importance of reliable and efficient gene modification confirmation will only increase, enabling researchers to have confidence in their experimental results and the integrity of their genetic manipulations.

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