Genomic DNA Preparation 예비레포트 결과레포트

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1. DNA (Deoxyribonucleic Acid)

DNA는 자연계에 존재하는 2종류의 핵산 중 디옥시 리보오스를 함유한 것의 총칭을 말하며, 디옥시리보핵 산(Deoxyribonucleic Acid)의 약칭이다. DNA는 뉴클레오 타이드의 중합체인 2개의 긴 가닥이 서로 꼬여 있는 이중나선구조로 고분자 화합물이다. DNA의 기본 단위는 뉴클레오타이드이며, 뉴클레오타 이드는 염기, 당, 인산으로 구성된다.
2. DNA Extraction

DNA를 얻기 위해서는 먼저 세포를 파괴해야 한다. 세포를 파괴하는 방법에는 원심분리와 같은 물리적 방법과 화학물질을 이용해 세포벽과 세포막을 파괴하는 화학적 방법이 있다. 세포 내에 DNA 외에 단일가닥의 RNA도 존재하므로 RNase를 처리하여, RNA를 제거한다. 단백질과 DNA를 변성시키고 페놀/클로로폼 추출법을 이용하여 시료 속의 단백질을 제거한다. 마지막으로 Ethanol과 Isopropanol을 사용하여 DNA를 침전시키고 원심분리 후, 침전물을 얻어 적당한 완충 용액에 녹여준다.
3. Spectroscopy (Nano Drop)

자외선, 가시광선과 같은 특정 파장의 빛이 용액을 통과할 경우, 용액 속에 들어 있는 특정 물질의 양에 따라 흡수되어 복사선의 세기가 감소할 수 있다. 이러한 빛의 흡수 현상을 이용하여 용액 속에 들어있는 물질의 양을 정량할 수 있다. Nano Drop machine은 이 원리를 이용하여 DNA의 농도를 측정한다.
4. Lambert-Beer 법칙

A =log(I0/I)=-logT=흡광도 T=I/I0 I₀=시료에 들어가는 빛의 세기 I=시료에서 나오는 빛의 세기 d=물질의 두께 c=용질의 몰농도 ε=몰 흡광계수 (비례상수)
5. Silica 막에 DNA 결합 원리

Chaotropic salts의 존재 하에 DNA는 silica 막에 결합한다. Silica 막에 DNA가 결합하는 원리는 chaotropic salts의 높은 이온 강도가 silica 표면의 음전하 감소를 일으키고 silica 막의 표면 전하의 감소는 음전하를 띤 DNA와 음전하를 띤 silica 사이의 정전기적 반발력을 감소시킨다. 그리고 DNA의 음이온과 Silica 음이온 사이에서 salt bridge가 형성되어 붙게 된다.
6. 고상추출방법

고상추출방법은 액체에 용해되는 성질이나 용질의 친화도를 사용해 혼합물을 원하는 성분과 원하지 않는 성분을 분리하는 것을 말한다. 이번 실험에서는 silica 막과 DNA가 서로 흡착하는 경우로 불순물은 흡착하지 않아 폐기하고 silica 막에 붙은 DNA는 물을 통해 silica에서 떨어뜨려 수집하는 방식으로 불순물과 분리시킨다.
7. Absolute Ethanol의 역할

에탄올은 DNA를 둘러싸고 있는 용매 껍질을 제거하여 DNA가 침전되고 응집되도록 하고 DNA 용해도를 최소화 염이 용해되도록 하면서 DNA를 분리해주는 역할을 한다. 또한, DNA는 친수성이라 물에 잘 녹는 성질을 가지고 있는데 Washing buffer로 불순물을 제거할 때 불순물과 같이 DNA가 씻겨 내려가는 것을 방지해주는 역할을 한다.
8. Buffer의 역할

Binding buffer는 chaotropic salts를 공급해서 silica와 DNA를 잘 결합하게 해주는 역할을 하며 고염도 농도 상태를 만들어 준다. Washing buffer는 DNA가 떨어지지 않게 고염도 농도 상태를 유지하며 다른 불순물을 씻어주는 역할을 한다. Elution buffer는 Washing buffer로 처리한 후 남아있는 DNA를 아래로 침전시켜주는 역할을 한다.
9. Proteinase K의 역할

Proteinase K는 단백질분해효소로 DNA 정제과정에서 DNA를 분해할 수 있는 nuclease(핵산 분해효소)를 빠르게 비활성화 시킨다. 또한, 단백질을 분해할 수 있으므로 세포 용해물(조직, 세포 배양 세포)의 단백질 파괴 및 핵산을 방출할 때 사용한다.
10. 실험 결과 및 고찰

실험 결과 4개 샘플의 평균 260/230은 1.9325, 260/280은 1.9875, 농도는 31.5 ng/μL로 나왔다. 오차가 발생한 이유로는 피펫으로 피를 옮길 때 오염, Proteinase K 혼합 불량, 추출 DNA 보관 온도 등이 고려되었다. 실험 1과 4의 차이는 에탄올과 isopropanol 사용이었는데, 에탄올은 소량의 DNA 침전에, isopropanol은 많은 양의 샘플에 유용하다. 마지막 원심분리에서 하층액을 보관한 이유는 EA가 남은 DNA를 침전시켜주기 때문이다.

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1. DNA (Deoxyribonucleic Acid)

DNA, or deoxyribonucleic acid, is the fundamental genetic material that carries the instructions for the development and functioning of all living organisms. It is a remarkable molecule that stores and transmits genetic information, enabling the inheritance of traits from one generation to the next. DNA is composed of two complementary strands that form a double helix structure, with each strand containing a sequence of four chemical building blocks called nucleotides. The unique sequence of these nucleotides encodes the genetic instructions that guide the growth, development, and reproduction of cells. DNA is essential for life, as it plays a crucial role in the storage, replication, and expression of genetic information, making it a central focus of study in the fields of biology, genetics, and biotechnology.
2. DNA Extraction

DNA extraction is the process of isolating and purifying DNA from biological samples, such as cells, tissues, or organisms. This process is fundamental in various fields, including molecular biology, genetics, forensics, and biotechnology. The goal of DNA extraction is to obtain a pure and concentrated sample of DNA that can be used for further analysis, such as sequencing, amplification, or genetic manipulation. The basic steps involved in DNA extraction typically include cell lysis, removal of proteins and other cellular components, and precipitation or purification of the DNA. Different techniques, such as organic solvent extraction, silica-based column purification, or magnetic bead-based methods, can be employed depending on the sample type and the intended downstream applications. Efficient and reliable DNA extraction is crucial for ensuring the quality and integrity of the genetic material, which is essential for accurate and reliable results in a wide range of scientific and diagnostic applications.
3. Spectroscopy (Nano Drop)

Spectroscopy, particularly the Nano Drop technique, is a powerful analytical tool used to measure the concentration and purity of DNA, RNA, and protein samples. The Nano Drop is a specialized spectrophotometer that requires only a small volume of sample (typically 1-2 μL) to determine the concentration and assess the purity of the nucleic acid or protein. The principle behind the Nano Drop is the measurement of the absorbance of the sample at specific wavelengths, which is directly proportional to the concentration of the target molecule. This technique is highly valuable in various fields, such as molecular biology, genetics, and biochemistry, as it allows researchers to quickly and accurately quantify and assess the quality of their samples before proceeding with downstream applications, such as PCR, sequencing, or protein analysis. The Nano Drop's ability to provide rapid and precise measurements, while using minimal sample volumes, makes it an indispensable tool in modern scientific laboratories.
4. Silica Membrane DNA Binding Principle

The silica membrane DNA binding principle is a widely used technique in DNA extraction and purification. This method relies on the ability of DNA to selectively bind to silica-based materials, such as silica gel or silica membranes, under specific conditions. The binding of DNA to the silica surface is primarily driven by the interaction between the negatively charged phosphate groups in the DNA backbone and the positively charged silanol groups on the silica surface. Additionally, the presence of chaotropic salts, such as guanidinium hydrochloride or sodium iodide, helps to disrupt the hydrogen bonds and hydrophobic interactions that hold the DNA in its native conformation, allowing it to bind more efficiently to the silica. Once the DNA is bound to the silica membrane, contaminants, such as proteins, salts, and other cellular components, can be washed away, leaving behind a purified DNA sample that can be eluted using a low-ionic-strength buffer or water. This silica membrane-based DNA extraction method is widely used in various applications, including molecular biology, forensics, and diagnostic testing, due to its simplicity, efficiency, and ability to produce high-quality DNA samples.
5. Role of Absolute Ethanol

Absolute ethanol, or 100% ethanol, plays a crucial role in the DNA extraction and purification process. Ethanol is commonly used as a precipitating agent to concentrate and recover DNA from aqueous solutions. The mechanism behind this is that ethanol reduces the solubility of DNA, causing it to precipitate out of the solution. This is achieved by the ethanol molecules disrupting the hydration shell around the DNA, effectively dehydrating the DNA and reducing its solubility. The addition of absolute ethanol, typically in combination with a salt, such as sodium acetate or ammonium acetate, facilitates the precipitation of DNA by further reducing its solubility. The precipitated DNA can then be collected by centrifugation or filtration, and the supernatant containing contaminants can be discarded. The use of absolute ethanol is a critical step in many DNA extraction protocols, as it allows for the efficient recovery and purification of DNA from complex biological samples, ensuring the integrity and purity of the extracted genetic material for downstream applications.
6. Role of Proteinase K

Proteinase K is an essential enzyme used in DNA extraction and purification protocols. Its primary role is to degrade and remove proteins from the sample, which is a crucial step in obtaining pure and high-quality DNA. Proteinase K is a serine protease that exhibits broad substrate specificity, effectively cleaving peptide bonds in a wide range of proteins, including enzymes, structural proteins, and DNA-binding proteins. During the DNA extraction process, Proteinase K is typically used in combination with lysis buffers containing detergents, such as SDS or Triton X-100, to disrupt cell membranes and release the cellular contents, including the DNA and proteins. The presence of Proteinase K in the lysis step helps to degrade and solubilize the proteins, preventing them from interfering with the subsequent DNA purification steps. By removing these protein contaminants, Proteinase K ensures that the extracted DNA is free from protein-based inhibitors, which could otherwise interfere with downstream applications, such as PCR, restriction enzyme digestion, or DNA sequencing. The use of Proteinase K is a crucial step in many DNA extraction protocols, as it helps to produce high-quality, pure DNA samples that are suitable for a wide range of molecular biology and biotechnology applications. Its ability to efficiently remove proteins without compromising the integrity of the DNA makes Proteinase K an indispensable tool in the field of DNA extraction and purification.

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