분자생물학 및 유전공학의 실험방법

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1. Restriction endonuclease

DNA의 specific short sequences를 인식하고 쪼갤 수 있는 enzyme입니다. Cloning vectors는 host cell에 외래 DNA를 삽입하기 위해 사용할 수 있는 DNA로, selectable markers와 replication origins을 가지고 있습니다.
2. Nucleases

Nucleases(핵산가수분해효소)는 phospho diester bond의 ester bond를 Hydrolyze하는 enzyme이며, Phosphatases(인산분해효소)는 a phospho mono ester bond의 ester bond를 Hydrolyze하는 enzyme입니다. Endonuclease(핵산내부가수분해효소)는 nucleic acid chain 내부의 bonds를 절단하는 enzyme이며, Exonuclease(핵산외부가수분해효소)는 end of a polynucleotide chain 특이적인 절단 enzyme입니다.
3. Cloning

Cloning은 a fragment of DNA를 requires a specially engineered vector로 수행하며, Recombinant DNA(재조합 DNA)는 artificial DNA molecule created by joining two (or more) DNA molecules입니다. Subclone은 breaking a cloned fragment into smaller fragments for further cloning을 의미합니다. Vector는 insert 가능한 상태의 plasmid DNA로, MCS, Origin, Selection marker를 모두 가져야 합니다.
4. Cloning vectors

Cloning Vector는 특징이 다 달라서 상황에 따라 specific하게 사용됩니다. Shuttle vector는 Prokaryotic ↔ eukaryotic 상호간 이동이 가능하며, expression vector는 유전자 발현을 유도하는 promoter를 포함하고, reporter gene은 형광 등으로 가시적으로 확인 가능한 gene입니다.
5. Nucleic acid detection

labeled nucleic acid를 complementary sequences에 Hybridization(혼성화)하면 혼합물에서 specific nucleic acids를 식별할 수 있습니다. Probe는 complementary fragment를 식별하는 labeled nucleic acid이며, Autoradiography는 방사선 probe를 이용해 image를 capture하여 원하는 nucleic acid를 식별합니다.
6. DNA separation techniques

Gel electrophoresis는 DNA fragments를 size별로 분리하며, density gradient centrifugation은 G-C content에 따른 밀도 차이를 이용하여 분리합니다.
7. DNA sequencing

Chain-termination sequencing은 ddNTPs(Dideoxynucleoside triphosphates)를 사용하여 DNA synthesis를 종결시키는 방법입니다. Primer는 DNA polymerase가 합성을 시작할 수 있는 3'-OH end를 제공합니다. 형광 ddNTPs와 capillary gel electrophoresis를 이용한 방법은 automated, high-throughput DNA sequencing을 가능하게 합니다.
8. PCR and RT-PCR

Polymerase chain reaction (PCR)은 a desired sequence를 증폭하며, RT-PCR은 reverse transcriptase를 이용해 RNA를 DNA로 convert하여 transcription level을 check하는데 주로 사용됩니다. Real-time, or quantitative, PCR은 특정 gene을 targeting할 수 있는 probe가 존재하여 FRET 반응을 통해 생성중인 DNA를 detection합니다.
9. Blotting methods

Southern blotting은 DNA를, Northern blotting은 RNA를, Western blotting은 단백질을 membrane에 transfer하여 detection하는 방법입니다. Epitope tag는 항체에 의해 확인될 수 있는 단백질에 첨가된 polypeptide입니다.
10. DNA microarrays

DNA microarrays는 작은 chip에 DNA sequences를 부착시켜 색상 농도차에 의해 gene expression을 확인하는 방법입니다. SNP(Single Nucleotide Polymorphism)는 DNA 서열에서 단일 염기 서열의 다양성을 의미합니다.
11. Gene Knockouts, Transgenics, and Genome Editing

Transgenic은 germline에 exogenous DNA를 도입하여 만들어진 생물체이며, knockouts는 유전자의 기능을 연구하기 위해 특정 sequence의 기능을 하지 못하게 하는 것입니다. Homologous recombination, Cre-lox system, Meganucleases, ZFNs, TALENs, CRISPR/Cas9 등의 다양한 gene editing 기술이 있습니다.

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1. Restriction endonuclease

Restriction endonucleases are enzymes that can recognize and cleave specific DNA sequences, known as restriction sites. These enzymes play a crucial role in molecular biology and genetic engineering, as they allow for the precise manipulation and analysis of DNA molecules. They are essential tools in techniques such as cloning, DNA sequencing, and genetic modification. Restriction endonucleases enable researchers to cut DNA at specific locations, facilitating the isolation, purification, and recombination of genetic material. This ability to precisely target and cleave DNA has been instrumental in advancing our understanding of gene structure, function, and regulation, as well as in the development of various biotechnological applications, including the production of genetically modified organisms, diagnostic tools, and therapeutic interventions. The continued study and utilization of restriction endonucleases remain a vital aspect of modern molecular biology and biotechnology.
2. Nucleases

Nucleases are a diverse group of enzymes that catalyze the hydrolysis of the phosphodiester bonds within nucleic acids, such as DNA and RNA. These enzymes play crucial roles in various biological processes, including DNA repair, gene expression regulation, and immune system function. Nucleases can be classified into different types based on their specificity and mode of action, such as exonucleases, which cleave nucleic acids from the ends, and endonucleases, which cleave internal bonds within the molecule. The study and application of nucleases have been instrumental in advancing our understanding of genetic mechanisms and enabling the development of numerous biotechnological tools and techniques. For example, restriction endonucleases, a subclass of nucleases, have revolutionized the field of genetic engineering by allowing for the precise manipulation and analysis of DNA. Additionally, nucleases have found applications in areas such as gene editing, diagnostic assays, and therapeutic interventions. As our understanding of nucleases continues to evolve, their potential for further advancements in molecular biology, biotechnology, and medicine remains immense.
3. Cloning

Cloning is the process of creating genetically identical copies of a biological entity, such as a cell, tissue, or organism. This powerful technique has had a profound impact on various fields, including biology, medicine, and agriculture. In the context of molecular biology, cloning has enabled researchers to isolate and amplify specific genes or DNA sequences, facilitating the study of genetic mechanisms, the production of recombinant proteins, and the development of genetically modified organisms. Cloning has also played a crucial role in the advancement of stem cell research, where it has been used to generate embryonic stem cells and to explore their potential for regenerative medicine. While the ethical and social implications of cloning have been widely debated, the scientific and technological advancements enabled by cloning have been instrumental in expanding our understanding of biological systems and driving progress in fields such as genetics, biotechnology, and regenerative medicine. As the field of cloning continues to evolve, it is likely to yield even more transformative applications in the years to come.
4. Cloning vectors

Cloning vectors are DNA molecules, such as plasmids or viruses, that are used to carry and propagate foreign DNA sequences in host cells, typically bacteria or yeast. These vectors act as vehicles for the insertion, replication, and expression of genetic material, enabling researchers to manipulate and study genes of interest. Cloning vectors possess essential features, such as origins of replication, selectable markers, and multiple cloning sites, which facilitate the efficient cloning and amplification of DNA fragments. The development and refinement of cloning vectors have been crucial in the advancement of genetic engineering, allowing for the creation of recombinant DNA, the production of therapeutic proteins, and the generation of genetically modified organisms. As the field of molecular biology has progressed, the diversity and capabilities of cloning vectors have expanded, with the introduction of specialized vectors for various applications, such as protein expression, gene silencing, and genome editing. The continued innovation and application of cloning vectors remain essential for driving advancements in biotechnology, medicine, and our understanding of biological systems.
5. Nucleic acid detection

Nucleic acid detection refers to the techniques and technologies used to identify, quantify, and analyze the presence and characteristics of DNA and RNA molecules. These methods are fundamental to various fields, including molecular biology, genetics, diagnostics, and forensics. Nucleic acid detection techniques, such as PCR (Polymerase Chain Reaction), hybridization assays, and sequencing, have revolutionized our ability to detect and study genetic material with unprecedented sensitivity and specificity. These tools have enabled researchers to investigate gene expression patterns, identify genetic variations, detect pathogens, and diagnose genetic disorders. Moreover, advancements in nucleic acid detection have facilitated the development of personalized medicine, where genetic information is used to tailor treatments and interventions to individual patients. As our understanding of the human genome and other complex biological systems continues to grow, the importance of reliable and efficient nucleic acid detection methods will only increase, driving further innovations in areas like disease diagnosis, forensic analysis, and environmental monitoring.
6. DNA separation techniques

DNA separation techniques are essential tools in molecular biology and genetics, allowing researchers to isolate, purify, and analyze specific DNA molecules or fragments. These techniques leverage the unique physical and chemical properties of DNA, such as size, charge, and sequence, to separate and fractionate DNA samples. Common DNA separation methods include gel electrophoresis, which separates DNA based on size, and chromatography techniques, such as ion-exchange and affinity chromatography, which exploit differences in charge and binding properties. The ability to effectively separate and purify DNA has been crucial for a wide range of applications, including genetic analysis, DNA sequencing, cloning, and the development of diagnostic assays. As the field of molecular biology has advanced, the repertoire of DNA separation techniques has expanded, with the introduction of more sensitive, efficient, and automated methods. These advancements have enabled researchers to tackle increasingly complex biological questions and have contributed to significant breakthroughs in areas like genomics, personalized medicine, and biotechnology.
7. DNA sequencing

DNA sequencing is the process of determining the precise order of nucleotides (A, T, C, and G) within a DNA molecule. This fundamental technique has revolutionized the field of molecular biology and has had a profound impact on our understanding of genetics, evolution, and the diversity of life. DNA sequencing has enabled researchers to decipher the genetic code of organisms, from simple bacteria to complex multicellular life forms, including humans. The development of increasingly efficient and cost-effective sequencing technologies, such as Sanger sequencing and next-generation sequencing, has facilitated the rapid and widespread acquisition of genetic information. This wealth of sequence data has fueled advancements in areas like genome assembly, comparative genomics, and the identification of genetic variations associated with diseases. DNA sequencing has also enabled the development of personalized medicine, where genetic information is used to tailor treatments and interventions to individual patients. As the field of DNA sequencing continues to evolve, with the emergence of long-read and single-molecule sequencing technologies, the potential for further breakthroughs in biology, medicine, and biotechnology remains immense.
8. PCR and RT-PCR

PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription-PCR) are powerful molecular biology techniques that have revolutionized the field of genetic analysis and diagnostics. PCR is a method for amplifying specific DNA sequences, allowing researchers to generate millions or billions of copies from a small initial sample. This technique has become indispensable in a wide range of applications, including genetic engineering, forensics, disease diagnosis, and environmental monitoring. RT-PCR, on the other hand, is a variation of PCR that is used to detect and quantify RNA molecules, enabling the study of gene expression and the identification of RNA-based biomarkers. The development and refinement of these techniques have been instrumental in advancing our understanding of genetic mechanisms, facilitating the diagnosis and monitoring of infectious diseases, and enabling the detection of genetic alterations associated with various disorders. As technology continues to evolve, with the introduction of more sensitive, rapid, and automated PCR and RT-PCR platforms, the potential applications of these techniques will continue to expand, driving further advancements in fields such as personalized medicine, biotechnology, and environmental science.
9. Blotting methods

Blotting methods, such as Southern blotting, Northern blotting, and Western blotting, are essential techniques in molecular biology and biochemistry for the detection and analysis of specific nucleic acids (DNA and RNA) and proteins, respectively. These methods involve the transfer of separated biomolecules from a gel or membrane to a solid support, followed by the use of labeled probes or antibodies to identify and quantify the target molecules. Blotting techniques have been instrumental in advancing our understanding of gene expression, protein structure and function, and the complex interactions within biological systems. Southern blotting, for example, has enabled the identification and characterization of specific DNA sequences, while Northern blotting has provided insights into RNA expression patterns. Western blotting, on the other hand, has become a widely used tool for the detection and quantification of proteins, facilitating the study of protein expression, post-translational modifications, and interactions. As the field of molecular biology has progressed, the sensitivity, specificity, and automation of blotting methods have improved, allowing for more accurate and efficient analysis of biomolecules. The continued development and application of blotting techniques will undoubtedly contribute to further advancements in areas such as diagnostics, drug discovery, and the understanding of complex biological processes.
10. DNA microarrays

DNA microarrays are high-throughput technologies that enable the simultaneous analysis of thousands or even millions of DNA sequences or gene expression levels in a single experiment. These platforms consist of miniaturized arrays of DNA probes, each designed to hybridize with a specific target sequence, allowing for the comprehensive profiling of genetic information. DNA microarrays have revolutionized the field of genomics, providing researchers with the ability to study gene expression patterns, identify genetic variations, and detect the presence of specific DNA sequences, including those associated with diseases. The applications of DNA microarrays span a wide range of areas, including disease diagnosis, drug discovery, pharmacogenomics, and environmental monitoring. As the technology has evolved, the resolution, sensitivity, and data analysis capabilities of DNA microarrays have improved, enabling more accurate and comprehensive insights into complex biological systems. The continued development and application of DNA microarray technology will undoubtedly contribute to further advancements in personalized medicine, the understanding of disease mechanisms, and the exploration of the genetic basis of life.
11. Gene Knockouts, Transgenics, and Genome Editing

Gene knockouts, transgenics, and genome editing are powerful tools in molecular biology and genetics that have transformed our ability to study and manipulate genetic information. Gene knockouts involve the targeted inactivation or removal of a specific gene, allowing researchers to investigate the function and importance of that gene within a biological system. Transgenics, on the other hand, involve the introduction of foreign genetic material into an organism, enabling the study of gene expression and the generation of genetically modified organisms. More recently, the development of genome editing technologies, such as CRISPR-Cas9, has revolutionized the field by providing a precise and efficient means of modifying DNA sequences, opening up new possibilities for gene therapy, disease modeling, and the creation of novel organisms with desired traits. These techniques have been instrumental in advancing our understanding of gene function, genetic mechanisms, and the genetic basis of disease, as well as in the development of innovative applications in fields like medicine, agriculture, and biotechnology. As the field continues to evolve, with the emergence of more advanced and versatile genome editing tools, the potential for transformative breakthroughs in our understanding and manipulation of genetic information remains immense.

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