[생명공학 레포트] gDNA isolation 및 T7E1 assay를 통한 genome editing 확인

문서 내 토픽

• 1. gDNA isolation

  gDNA는 plasmids와 같은 extra-chromosomal DNAs와는 다르게 chromosomal DNA를 의미한다. gDNA를 분리하는 단계에서는 cell과 CLS(Cell Lysis Solution), proteinase K를 섞어 DNA 추출을 위해 cell을 파괴하는 역할을 한다. CLS에는 SDS, Tris-HCl, EDTA 등이 들어가 있으며, SDS는 cell membrane의 lipid를 제거하고, Tris-HCl은 pH 변화를 방지하며, EDTA는 세포 내의 DNases의 역할을 억제한다. proteinase K는 DNA를 분해할 수 있는 단백질을 분해시켜준다. 이후 PPS(protein precipitation solution)을 넣어 단백질을 침전시키고, isopropanol을 넣어 DNA를 침전시킨다. 마지막으로 에탄올로 washing하여 순수한 DNA를 얻어낸다.

• 2. PCR (Polymerase Chain Reaction)

  PCR은 관심 있는 DNA 서열을 수백만 배로 증폭시키는 기술이다. PCR에 필요한 재료에는 DNA template, primers(forward, reverse), DNA polymerase, dNTPs 등이 있다. PCR 과정은 Initialization, Denaturation, Annealing, Elongation 단계로 진행되며, 이를 35-40회 반복하여 DNA를 증폭시킨다.

• 3. T7E1 assay

  T7E1은 endonuclease의 일종으로, CRISPR/Cas9 등으로 genome editing을 실시한 DNA에서 genome editing이 제대로 되었는지 확인하는 데 사용된다. T7E1은 heteroduplexed DNA의 구조적인 deformities를 감지하고 절단하는 능력이 있다. PCR로 증폭된 product에 T7E1을 처리하면 editing된 부분이 절단되어 전기영동 시 최소 2개 이상의 선이 나타나게 된다. 이를 통해 Genome Editing이 성공적으로 이루어졌는지 확인할 수 있다.

• 4. Next Generation Sequencing (NGS)

  기존의 Sanger sequencing 방법은 효율적이지 못한 한계가 있었다. NGS 기술은 이를 극복하고 좀 더 효율적인 DNA 서열분석 방법을 제공한다. 대표적인 NGS 방법인 Illumina sequencing에서는 DNA fragments에 adaptors를 붙이고, flow cell에 부착시켜 PCR로 증폭한 후, 염기 종류에 따라 다른 빛을 내는 형광 물질이 결합된 dNTP를 활용하여 서열을 분석한다. NGS 기술은 diagnostic virology 분야에서 새로운 바이러스 식별, human virome 연구, full-length viral genome sequencing, virus-host interaction 연구 등에 활용되고 있다.

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• 1. gDNA isolation

  gDNA (genomic DNA) isolation is a fundamental technique in molecular biology and genetics. It involves extracting and purifying the complete genetic material from cells or tissues. This process is crucial for various downstream applications, such as PCR, sequencing, and genetic analysis. The ability to obtain high-quality, intact gDNA is essential for accurate and reliable results in these experiments. gDNA isolation methods typically involve cell lysis, protein denaturation, and selective precipitation or column-based purification to remove contaminants like RNA, proteins, and other cellular components. The choice of isolation method depends on the sample type, the desired purity and yield, and the intended downstream applications. Proper gDNA isolation is a critical step that can significantly impact the success and quality of subsequent molecular biology experiments.

• 2. PCR (Polymerase Chain Reaction)

  PCR (Polymerase Chain Reaction) is a revolutionary technique in molecular biology that allows for the exponential amplification of specific DNA sequences. This powerful tool has transformed various fields, including genetics, diagnostics, forensics, and biotechnology. PCR enables researchers to generate millions or even billions of copies of a target DNA fragment from a small initial sample, making it possible to study and analyze genetic material in great detail. The process involves thermal cycling, where the DNA is repeatedly denatured, annealed with specific primers, and extended by a DNA polymerase enzyme. The ability to selectively amplify DNA sequences has led to numerous applications, such as disease diagnosis, genetic profiling, pathogen detection, and DNA sequencing. PCR has become an indispensable technique in modern molecular biology, allowing for sensitive, specific, and efficient analysis of genetic information.

• 3. T7E1 assay

  The T7E1 (T7 Endonuclease I) assay is a widely used method for detecting and analyzing genome editing events, particularly those involving CRISPR/Cas9 technology. This assay relies on the ability of the T7E1 enzyme to recognize and cleave mismatched DNA duplexes, which can arise from the introduction of insertions, deletions, or other modifications in the target DNA sequence. By amplifying the target region and subjecting the PCR product to T7E1 digestion, researchers can identify the presence and frequency of genome editing events in a sample. The T7E1 assay is a valuable tool for screening and validating the efficiency of CRISPR/Cas9 systems, as well as for monitoring the outcomes of genome editing experiments. It provides a simple, cost-effective, and sensitive method to assess the success of genome engineering efforts, making it an essential technique in the field of gene editing and genome manipulation.

• 4. Next Generation Sequencing (NGS)

  Next Generation Sequencing (NGS), also known as high-throughput sequencing, is a transformative technology that has revolutionized the field of genomics and molecular biology. NGS platforms enable the rapid and cost-effective sequencing of entire genomes, transcriptomes, or targeted regions of interest, providing unprecedented insights into the genetic makeup of organisms. This powerful technology has enabled researchers to explore the complexity of genomes, identify genetic variations, detect novel mutations, and unravel the molecular mechanisms underlying various biological processes and diseases. NGS has significantly accelerated the pace of scientific discoveries, allowing for the generation of vast amounts of sequence data that can be analyzed and interpreted to gain a deeper understanding of biological systems. The versatility of NGS applications, ranging from whole-genome sequencing and targeted gene panels to metagenomics and single-cell analysis, has made it an indispensable tool in fields such as genetics, genomics, personalized medicine, evolutionary biology, and environmental microbiology. As the technology continues to evolve, NGS is poised to unlock even more transformative insights and drive further advancements in our understanding of the living world.