獸醫病毒學實習五:DNA 的萃取

(一) 目的:

1. 學習 DNA 的各種萃取方式。 2. 了解萃取 DNA 時的注意事項。 3. 了解 kit 套組中使用試劑之效果。 4. 以肉眼觀察 DNA 萃取液的形態。 5. 實際操作同時萃取出病毒與細胞的 DNA。 6. 儀器與設備: 1. 水浴槽 2. 離心機 4. 廢液桶 5. 微量分注管(pipette) 6. 塑膠尖管(tips) 7. Column Kit 8. 離心管 9. 培養皿 10.手套 11. Tray 12.保麗龍盒 13.冰塊 14. boat 15. Cap 16. PBS 溶液 17. 培養皿內含假性狂犬病病毒、被感染的 PK-15 細胞與少量維持性培養液 18. Kit 內含試劑: Buffer C-L, RNase A, Proteinase K, Buffer P-D, Buffer W1 和 Buffer W2 19. DDW(二次蒸餾水)

●column 下半部 ●column 上半部 ●kit (三) 步驟 1. 戴上手套，並以酒精消毒雙手，並將紙巾以酒精噴濕後放置在桌上 2. 取一含有假性狂犬病病毒、被感染之細胞與少許培養液之培養皿

●含有假性狂犬病病毒、被感染之細胞與少許培養液之培養皿 3. 打開培養皿，並將培養皿略為傾斜，用微量分注器 pipette 將培養液吸盡並置 於廢液桶(直接倒掉會使培養液殘留在培養皿邊緣,且對細胞刺激太大)

●以 pipette 吸取掉培養皿中所有的培養液

4. 將藍色 pipette 刻度轉成 035，裝上 tip 之後，吸取 PBS 350 μl 並注入培養皿中 ●吸取 PBS 350 μl 並注入培養皿

5. 自 kit 中拿出 Buffer C-L,並將藍色 pipette 刻度轉成 015，裝上 tip 之後，吸取 Buffer C-L 150 μl 並注入培養皿中(Buffer C-L 類似清潔劑)

●吸取 Buffer C-L 150 μl 並注入培養皿

6. 將培養皿拿起輕輕搖晃，使細胞被消化下來 ●輕輕晃動培養皿

7. 取一離心管，並將事先將水浴槽溫度調至 56°C，使其溫度慢慢上升 8. 將藍色 pipette 刻度轉成 050，裝上 tip 之後，吸取培養皿中所有溶液並注入 離心管中 (可觀察到溶液呈現黏稠狀，因其即為 DNA)

●吸取培養皿中所有溶液並注入離心管中 ●置於 tray 上的離心管 9. 取一黃色的 pipette，將刻度轉成 008，裝上 tip 之後，吸取 0.8 µl 的 RNase A 並注入離心管中

●吸取 0.8 µl 的 RNase A 注入離心管中 10. 將黃色的 pipette 刻度轉成 080，裝上 tip 之

後，吸取 8 µl 的 proteinase K 並注入離心管中 ●吸取 8 µl proteinase K 的注入離心管中

11. 將離心管拿在手上拍彈數次(為使其混合均勻) ●將離心管拿在手上拍彈數次

12. 將離心管開口處套上 cap(以防止水浴時水跑入離心管內) ●將離心管開口處套上 cap

13. 將加蓋的離心管套入 boat 中，放入 56°C 水浴槽內，使其水浴十五分鐘以上 ●加蓋離心管放進 boat 後開始水浴

14. 十五分鐘過後取出離心管，(水浴時間超過十五分鐘沒關係)拆去 boat 和 cap

15. 將藍色 pipette 刻度轉成 035，裝上 tip 之後,吸取 360 µl Buffer P-D 並注 入離心管中(剛加入會有明顯分層現象)

●左:將 360 µl Buffer P-D 並注入離心管 ●右:分層現象 16. 將離心管拿在手

上上下翻轉三十秒使

其均勻混合 ●將離心管上下搖晃三十秒

17. 在室溫下離心機以 13000 rpm 離心 10 分鐘後，將細胞碎片離心下來 ●上層上清液含 DNA;下層沉澱物為細胞碎片 18. 將 column 上半部與下半部組裝起來，以 pipette 吸取

上清液注入 column (column 上的膜可吸附 DNA) ●上清液注入 column 19. 將 column 放入離心機 並以 13000 rpm 離心 1 分

鐘。

20. 將離心下來的液體丟棄。重新將 column 組裝好 ●離心完後 column 內的液體倒掉 21. 將藍色pipette刻度轉成050，並吸取500 µl的Buffer

W1 到 column 後，放入離心機以 13000 rpm 離心 1 分鐘 ●取 500 µl的Buffer W1到 column 並拿去離心一分鐘

22. 將離心下來的液體丟棄。重新將 column 組裝好 23. 將藍色 pipette 刻度轉成 070，並吸取 700 µl 的 Buffer W2 到 column 後， 放入離心機以 13000 rpm 離心 1 分鐘 24. 將離心下來的液體丟棄。重新將 column 組裝好 25. 直接將 column 放入離心機以 13000 rpm 離心 1 分鐘 26. 將 column 上半部組裝到 1.5 ml 的離心管上，以 pipette 吸取 100 µl 的 DDW 加到膜中央後作用 1 分鐘 27. 最後以 13,000 rpm 離心 1 分鐘(不用蓋上離心管蓋子)， 28. 離心完成後，將 column 上半部丟棄，蓋上離心管蓋子，即可得到 DNA

●凝膠狀液體即是萃取得到的 DNA

(四) 補充資料 :

1. 核酸萃取方法:

萃取純化 DNA 的方法很多，目前最常見的方法分成兩類：管柱萃取純化法

（Column Purification）與試劑萃取純化法（Reagents Purification）。在試劑萃取

純化法方面，又分成兩類：有機溶劑萃取法與非有機溶劑萃取法。每種萃取方法

各有其優點、缺點，然而，由於有機溶劑具腐蝕毒性，人員在操作安全上需特別

小心，同時其廢棄物的處理亦較為煩瑣，因此，使用非有機溶劑萃取方法已慢慢

成為趨勢。

A. 管柱萃取純化法(Column Purification)：其原理主要是先以陰離子性清潔劑將

細胞打破，並使蛋白質變性，以抑制 DNase 的活性，隨後加入蛋白酶

K(Proteinase K)讓 DNA 上的蛋白質解離，以增加 DNA 產率。接著將此含有 DNA

的混合物滴入純化管柱中，DNA 會與純化管柱中的過濾膜結合，其它物質不會

與過濾膜結合而流出，最後再利用緩衝液將過濾膜上的 DNA 洗提使其流出。

B. 有機溶劑萃取純化法(Organic Sorvant Reagents Purification)：其原理主要是利

用有機溶劑：酚(Phenol)、氯仿(Chloroform)將細胞溶解，經過超高速離心，再利

用 DNA 會分佈於水相層的原理，將 DNA 分離出來，經過酒精清洗沉澱，最後

回溶於緩衝液中。

C. 非有機溶劑萃取純化法(Non-Organic Sorvant Reagents Purification)：其原理主

要是先以陰離子性清潔劑將細胞打破，並使蛋白質變性，以抑制 DNase 的活性，

並加入蛋白酶 K(Proteinase K)讓 DNA 上的蛋白質解離，以增加 DNA 產率。接

著再以鹽類沉澱方法去除蛋白質。萃取到的 DNA 以酒精沉澱法濃縮，並回溶於

緩衝溶液中保留。完成 DNA 萃取與純化後,需要進一步作品質上定性及定量的

控制分析(Quality Control Assay)，包括：瓊膠電泳分析(Agarose Gel Electrophoresis

Analysis)與紫外光吸光值分析(UV Absorption Analysis)。

2. 核酸分析方法:

A. 瓊膠電泳分析：利用 DNA 分子大小與帶負電荷與的特性，當其通過洋菜瓊

膠所形成的孔篩時，DNA 會往陽極方向移動，分子越小移動速度越快，分子越

大移動速度越慢。如果檢體組織細胞中的 DNA 分子完整無缺，未被破壞，利用

上述之技術方法分離純化所得的 DNA 分子大小大約為 23000 bp，透過此項檢測

可以得知 DNA 是否保持完整、或是有被分解的現象。

B. 紫外光吸光值分析：利用 DNA 分子在波長 260nm 時有吸光、蛋白質在波長

280nm 有吸光的特性，分析 DNA 的濃度與純度。當波長 260nm 的吸光值為 1

時，DNA 的濃度為 50ug/ml，依此類推，若波長 260nm 的吸光值為 0.5 時，DNA

的濃度則為 25ug/mL。DNA 的純度可利用波長 260nm/波長 280nm 的比值來評

估，當 A260/A280 的比值越大，代表蛋白質汙染越低、DNA 純度越高。當

A260/A280 的比值介於 1.7~2.0 時，則表示此 DNA 的品質相當高！若低於此

值，表示蛋白質未被去除乾淨。

3. DNA 的結構:

Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. DNA is a nucleic acid; alongside proteins and carbohydrates, nucleic acids compose the three major macromolecules essential for all known forms of life. Most DNA molecules are double-stranded helices, consisting of two long biopolymers made of simpler units called nucleotides—each nucleotide is composed of a nucleobase (guanine, adenine, thymine, and cytosine), recorded using the letters G, A, T, and C, as well as a backbone made of alternating sugars (deoxyribose) and phosphate groups (related to phosphoric acid), with the nucleobases (G, A, T, C) attached to the sugars. DNA is well-suited for biological information storage, since the DNA backbone is resistant to cleavage and the double-stranded structure provides the molecule with a built-in duplicate of the encoded information. The two strands of DNA run in opposite directions to each other and are therefore anti-parallel, one backbone being 3′ (three prime) and the other 5′ (five prime). This refers to the direction the 3rd and 5th carbon on the sugar molecule is facing. Attached to each sugar is one of four types of

molecules called nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes genetic information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription. Within cells, DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. The obsolete synonym "desoxyribonucleic acid" may occasionally be encountered, for example, in pre-1953 genetics. DNA is a long polymer made from repeating units called nucleotides.DNA was first identified and isolated by Friedrich Miescher and the double helix structure of DNA was first discovered by James Watson and Francis Crick. The structure of DNA of all species comprises two helical chains each coiled round the same axis, and each with a pitch of 34 ångströms (3.4 nanometres) and a radius of 10 ångströms (1.0 nanometres). According to another study, when measured in a particular solution, the DNA chain measured 22 to 26 ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit measured 3.3 Å (0.33 nm) long.Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, consists of approximately 220 million base pairs and is 85 mm long. In living organisms DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, which interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. A polymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide. The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form

phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA. A section of DNA. The bases lie horizontally between the two spiraling strands. (animated version). The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. In the aqueous environment of the cell, the conjugated π bonds of nucleotide bases align perpendicular to the axis of the DNA molecule, minimizing their interaction with the solvation shell and therefore, the Gibbs free energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate to form the complete nucleotide, as shown for adenosine monophosphate. 4. DNA condensation DNA condensation refers to the process of compacting DNA molecules in vitro or in vivo. Mechanistic details of DNA packing are essential for its functioning in the process of gene regulation in living systems. Condensed DNA often has surprising properties, which one would not predict from classical concepts of dilute solutions. Therefore DNA condensation in vitro serves as a model system for many processes of physics, biochemistry and biology.In addition, DNA condensation has many potential applications in medicine and biotechnology. DNA diameter is about 2 nm, while the length of a stretched single molecule may be up to several dozens of centimetres depending on the organism. Many features of the DNA double helix contribute to its large stiffness, including the mechanical properties of the sugar-phosphate backbone, electrostatic repulsion between phosphates (DNA bears on average one elementary negative charge per each 0.17 nm of the double helix), stacking interactions between the bases of each individual strand, and strand-strand interactions. DNA is one of the stiffest natural polymers, yet it is also one of the longest molecules. This means that at large distances DNA can be considered as a flexible rope, and on a short scale as a stiff rod. Like a garden hose, unpacked DNA would randomly occupy a much larger volume than when it is orderly

packed. Mathematically, for a non-interacting flexible chain randomly diffusing in 3D, the end-to-end distance would scale as a square root of the polymer length. For real polymers such as DNA this gives only very rough estimate; what is important, is that the space available for the DNA in vivo is much smaller than the space that it would occupy in the case of a free diffusion in the solution. In order to cope with the volume constraints, DNA has a striking property to pack itself in the appropriate solution conditions with the help of ions and other molecules. Usually, DNA condensation is defined as "the collapse of extended DNA chains into compact, orderly particles containing only one or a few molecules".[3] This definition applies to many situations in vitro and is also close to the definition of DNA condensation in bacteria as "adoption of relatively concentrated, compact state occupying a fraction of the volume available". In eukaryotes, the DNA size and the number of other participating players are much larger, and a DNA molecule forms millions of ordered nucleoprotein particles, the nucleosomes, which is just the first of many levels of DNA packing. 5. DNA condensation in viruses In viruses and bacteriophages, the DNA or RNA is surrounded by a protein capsid, sometimes further enveloped by a lipid membrane. Double-stranded DNA is stored inside the capsid in the form of a spool, which can have different types of coiling leading to different types of liquid-crystalline packing. This packing can change from hexagonal to cholesteric to isotropic at different stages of the phage functioning. Although the double helices are always locally aligned, the DNA inside viruses does not represent real liquid crystals, because it lacks fluidity. On the other hand, DNA condensed in vitro, e.g. with the help of polyamines which are also present in viruses, is both locally ordered and fluid. 6. RNase A RNase A is a relatively small protein (124 residues, ~13.7 kDa). It can be characterized as a two-layer \alpha + \beta protein that is folded in half to resemble a taco, with a deep cleft for binding the RNA substrate. The first layer is composed of three alpha helices (residues 3-13, 24-34 and 50-60) from the N-terminal half of the protein. The second layer consist of three β-hairpins (residues 61-74, 79-104 and 105-124 from the C-terminal half) arranged in two β-sheets. The hairpins 61-74 and 105-124 form a four-stranded, antiparallel β-sheet that lies on helix 3 (residues 50-60). The longest β-hairpin 79-104 mates with a short β-strand (residues 42-45) to form a three-stranded, antiparallel β-sheet that lies on helix 2 (residues 24-34).

RNase A has four disulfide bonds in its native state: Cys26-Cys84, Cys58-110, Cys40-95 and Cys65-72. The first two (26-84 and 58-110) are essential for conformational folding; each joins an alpha helix of the first layer to a beta sheet of the second layer, forming a small hydrophobic core in its vicinity. The latter two disulfide bonds (40-95 and 65-72) are less essential for folding; either one can be reduced (but not both) without affecting the native structure under physiological conditions. These disulfide bonds connect loop segments and are relatively exposed to solvent. Interestingly, the 65-72 disulfide bond has an extraordinarily high propensity to form, significantly more than would be expected from its loop entropy, both as a peptide and in the full-length protein. This suggests that the 61-74 β-hairpin has a high propensity to fold conformationally. RNase A is a basic protein (pI = 9.63); its many positive charges are consistent with its binding to RNA (a poly-anion). More generally, RNase A is unusually polar or, rather, unusually lacking in hydrophobic groups, especially aliphatic ones. This may account for its need of four disulfide bonds to stabilize its structure. The low hydrophobic content may also serve to reduce the physical repulsion between highly charged groups (its own and those of its substrate RNA) and regions of low dielectric constant (the nonpolar residues). The N-terminal α-helix of RNase A (residues 3-13) is connected to the rest of RNase A by a flexible linker (residues 16-23). As shown by F. M. Richards, this linker may be cleaved by subtilisin between residues 20 and 21 without causing the N-terminal helix to dissociate from the rest of RNase A. The peptide-protein complex is called RNase S, the peptide (residues 1-20) is called the S-peptide and the remainder (residues 21-124) is called the S-protein. The dissociation constant of the S-peptide for the S-protein is roughly 30 pM; this tight binding can be exploited for protein purification by attaching the S-peptide to the protein of interest and passing a mixture over an affinity column with bound S-protein. [A smaller C-peptide (residues 1-13) also works. The RNase S model system has also been used for studying protein folding by coupling folding and association. The S-peptide was the first peptide from a native protein shown to have (flickering) secondary structure in isolation. RNase A cleaves specifically after pyrimidine nucleotides. Cleavage takes place in two steps: first, the 3’,5’-phosphodiester bond is cleaved to generate a 2’,3’-cyclic phosphodiester intermediate; second, the cyclic phosphodiester is hydrolyzed to a 3’-monophosphate. It can be inhibited by ribonuclease inhibitor protein, by heavy metal ions, and by uridine-vanadate complexes.The positive charges of RNase A lie mainly in a deep cleft between two lobes. The RNA substrate lies in this cleft and is cleaved by two catalytic histidine residues, His12 and His119, to form a 2',3'-cyclic phosphate

intermediate that is stabilized by nearby Lys41. RNase A protein may use the morpheein model of allosteric regulation. RNase A, and to a greater extent its oligomers and some homologs (such as onconase from frogs), have cytotoxic and cytostatic effects, particularly on cancer cells. This attribute has led to the development of onconase as a cancer therapeutic. As with many protein drugs from a non-human source, the internal use of non-human ribonucleases such as onconase is limited by the patient's immune response. Ribonuclease is also related to angiogenin, which is involved in blood vessel development. 7. proteinase K In molecular biology proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8. The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa). Activated by calcium (1–5 mM), the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digestion with Proteinase K for the purification of nucleic acids is usually performed in the presence of EDTA. Proteinase K is also stable over a wide pH range (4–12), with a pH optimum of pH 8.0. An elevation of the reaction temperature from 37 °C to 50–60 °C may increase the activity several times, like the addition of 0.5–1% sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) and urea (4 M). Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease-inhibitors AEBSF, PMSF or DFP inhibit the activity. Proteinase K will not be inhibited by Guanidinium chloride, Guanidinium thiocyanate, urea, Sarkosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or, interestingly, by other serine protease inhibitors like Nα-Tosyl-Lys Chloromethyl Ketone (TLCK) and

Nα-Tosyl-Phe Chloromethyl Ketone (TPCK). Protease K activity in commonly used buffers. Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. It is highly suited to this application since the enzyme is active in the presence of chemicals that denature proteins, such as SDS and urea, chelating agents such as EDTA, sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors. Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. Some examples for applications: Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5–1% SDS. Purification of genomic DNA from bacteria (miniprep): bacteria from a saturated liquid culture are lysed and proteins are removed by a digest with 100 μg/ml Proteinase K for 1 h at 37 °C; The enzyme's activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide substrates, denaturants inhibit the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease.

(Partly adapted from 維京百科)