DNA Extraction and Purification Isolation of DNA is often the first step before further analysis

Resources: Cell-eukaryotes-chromesome Bacteria-prokaryotes- chromesome/plasmids

This experiment fits well into exploring cell structures: Where is the DNA located? What properties of cell membranes allow us to break them open with detergents? Why is protease used to assist in extraction of DNA? Further discussion later in workshop.

Cell Structures Examples of cell types

DNA structure O Na+ P Base CH2 Sugar OH Graphic showing Na+ ions associating with negatively charged phosphate groups on DNA backbone.

The 4 Bases A pairs with T G pairs with C The bases pair up to form the rungs of the ladder. A pairs with T G pairs with C

DNA in the nucleus is packaged into chromosomes

Chromosomes come in pairs There are 46 chromosomes in each cell. (23 pairs)

plasmids

Preparing Cell samples Initial methods vary based on the type of sample being processed. Material soaked and cells are released into solution. Physical agitation may assist. Loose cells may be concentrated by centrifuging to pellet, and then remove supernatant.

General steps of Extraction “Conventional ” Technique 1 Cells are put into a solution (usually) And broken open to release contents, getting cellular material into solution. enzyme酶（溶菌酶，蛋白酶）, SDS

3 Cell extract is treated to remove all components except DNA*** 4 DNA solution is concentrated 5 Analysis of purified DNA

Plasmid extraction Features: small and circular independent DNA molecules 􀂄 take advantage of : smaller, more compact(致密)

Cells must be broken open to release contents 1st step: add Lysis Buffer Preparing Cell Extract Cells must be broken open to release contents

add lysis buffer to your tube If anyone ends up with a lot more than 5 mls (7 mls or more), they need to remove the excess and discard it. DNA precipitation may not occur optimally if much more than 5 mls of cell lysates are present when alcohol is added during the precipitation step. It’s important NOT to shake the lysed cells – this will shear the DNA (break it into small fragments) and shorn DNA will not precipitate well. Place the cap on the tube, and gently invert tube 5 times (don’t shake it!). Observe your tube, notice any changes:become clear

Principles : alkaline-lysis method碱裂解 2 When pH12.0～12.6, both liner chromesome and circular plasmids are denatured.—like wool ball/single strand ss 氢键断裂，双链解开 Chromesome are totally separated , but plasmids linked Principles : alkaline-lysis method碱裂解

3 When pH adjusted back to pH7.0, plasmids renature/reanneal rapidly, plasmids soluble可溶,but chromesome can’t.

notice any changes:become cloudy Adjust pH:溶液III, Slowly add cold乙酸钾溶液,pH=4.8, holding the tube at a 45°angle. gently invert it a few times. notice any changes:become cloudy Control time: Not too long

Centrifugation 13,500 rpm : chromesome , larger RNA and protein-SDS complex are precipitated, plasmids is left in supernatant. 质粒在上清中。

Still some RNA and protein left: Rnase and proteinase Centrifuge to remove insoluble cell debris Plasmid, RNA, protein chromexome,RNA protein Still some RNA and protein left: Rnase and proteinase Transfer supernatant to another tube

vary with different protocols 方案 purification Steps : vary with different protocols 方案 Solution 溶液:仅1种-- 5-6 种. 纯化:Ion-exchange resin column 过膜或过柱,或不需要—. 酚-氯仿抽提 Time needed: 20-45min Plasmid quality:high purity or ....

Elution of DNA. DNA is released from column DNA purification with silica column Resin columns load onto column. Nucleic acids bind to membrane and other components flow through Column washing. removes any contaminants Elution of DNA. DNA is released from column

Purification of DNA

Purification of DNA Add phenol or phenol/chloroform (1:1). Proteins are precipitated; form white layer at interface between organic and aqueous layers. Aqueous solution removed.

Precipitate DNA: Ice Cold Alcohol • DNA does not dissolve in alcohol. • The addition of cold alcohol makes the DNA clump together and precipitate out of solution. Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands.

Slowly add cold alcohol, holding the tube at a 45°angle. It is important to add the alcohol gently and keep tube at a 45 degree angle. After a few minutes, they should see small bubbles with DNA strands attached. If they see a nice clump of DNA, they can remove it to necklace vial before inversion. Alternatively, they can gently mix/tilt their test tube to see the DNA come out of solution and then make a necklace. It's important NOT to shake and dissociate the DNA that is precipitating.

Cap tube, gently tilt tube on its side then turn upright about 10 times until both the water and alcohol phases have mixed and the DNA comes out of solution. Let stand undisturbed for 5 min at room temperature.

Centrifuge to precipitate plasmid sometimes can’t see. Dry at air 5-10min， dissolve in 50-100ul wateror or 1xTE , according to concentration you need, generally 5-10ml bateria，add 50ul -100ul，plasmid 0.5-1ug/ul Store at: 4ºC (refrigeration), -20ºC (freezer), -70ºC (deep freezer). First, remove the plastic stopper from the vial (you will replace the stopper after transferring your DNA). Don’t lose the stopper! It’s small and hard to see. Next, using a disposable transfer pipet, carefully transfer the DNA strands to the glass amulet. Don’t overfill the amulet; the stopper will be difficult to seal on the vial if there is too much liquid inside.

Plasmid: alkaline-lysis method 溶液I: 50mmol/L葡萄糖，10mmol/L EDTA (pH8.0)， 25mmol/L Tris-HCl (pH8.0)； 溶液II: 0.2mol/L NaOH, 1%SDS,proteinase K, 溶液III: 乙酸钾溶液3M, pH=4.8 RNase A： 10mg/ml； Washing: chloroform氯仿, isoamyl异戊醇alcohol, and ethanol TEbuffere: Tris-HCl, EDTA，pH8.0；

Preparing Cell Extract: Chemical Methods Attack cell wall and cell membrane Cell wall = lysozyme, EDTA or both Cell membrane = detergent (SDS) 溶液I: 50mmol/L葡萄糖，10mmol/L EDTA (pH8.0)， 25mmol/L Tris-HCl (pH8.0)；proteinase K 溶液II: 0.2mol/L NaOH, 1%SDS EDTA binds Mg ions inhibits enzymes that can degrade DNA helps break down cell wall

Tris buffer to maintain the pH of the solution at a level where DNA is stable Sodium dodecyl sulphate (SDS) is a detergent; removes lipid molecules, cause cell and nuclear membrane disruption. O S - CH2 CH3 SDS Can talk about lipid bilayer model of cell membranes and how detergents work. SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage. Na+

Addition of:Protease,salt, RNase Protease :to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA. Protease treatment increases the amount of intact DNA extracted. Histones, which cause DNA to supercoil (compact) in the chromosome, are proteins.

Addition of:Protease, salt, RNase Salt: The protease solution already contains salt. Na+ ions of NaCI bind to the phosphate groups of DNA, neutralizing the electric charge of the DNA. The Na+ ions allows DNA molecules to come together instead of repelling each other, thus making it easier to renature when pH is neutral. RNase: Histones, which cause DNA to supercoil (compact) in the chromosome, are proteins.

Method can be adapted for large plamid If plasmids are large (40 - 100 kb) and occur in low copy numbers in cell. Traditional protocols, based upon variations of the standard alkaline-lysis method, which separates plasmid DNA from chromosomal DNA based upon its ability to rapidly reanneal after denaturation at high pH. typically difficult to isolate , with low yields of plasmid DNA.

Method can be adapted for large plamid For the rapid large-scale visualization of native plasmids. Rapid (under 1 hour), easy, very inexpensive and reliable From a variety of both Gram-positive and Gram-negative genera including Shigella, Klebsiella, E. coli, Pseudomonas, Bacillus, Streptococcus, Staphylococcus, and Enterococcus, as well as BACs from E. coli.

Method can be adapted for large plamid Commercial kits originally designed for the isolation of Bacterial Artificial Chromosomes (BACs) can be used. However, these are expensive and still require hours of sustained effort. New excellent protocol has been successfully modified for the isolation of large, low copy plasmids, with little or no contaminating chromosomal DNA.

lysis and alkaline denaturation steps are combined, proteins are removed via a simple ammonium acetate/chloroform step. DNA precipitated using a polyethylene glycol/NaCl step. No ethanol precipitation is required. If additional purification is required, extracted DNA can be further processed through a column .

Outline of plasmid extraction protocol 1. Grow strains in broth overnight 2. Pellet 1.5 - 2 ml of cells by centrifugation. Remove supernatant. 3. Resuspend by vortexing in 100 µL resuspension buffer (50 mM glucose/10 mM EDTA/10 mM Tris-Cl, pH 8.0).

Outline of plasmid extraction protocol 4. Add 200 µL lysis Mix by inversion. Incubate 5 minutes at room temperature. 5. Add 150 µL 7.5 M ammonium acetate and 150 µl chloroform. Mix by inversion. Chill on ice 10 minutes. Centrifuge 10 minutes.

Outline of plasmid extraction protocol 6. Transfer supernatant to 200 µL precipitation solution (30% polyethylene glycol 8000/1.5 M NaCl). Mix by inversion. Chill on ice 15 minutes.聚乙二醇 7. Centrifuge to pellet DNA. Remove supernatant. Resuspend in TE or water.

Notes on the protocol For Gram positives, incubation with fresh lysozyme added to the resuspension buffer is recommended. Step 2 This step both lyses the cells and denatures the DNA. For all post-lysis steps, mix by gentle inversion of the tube four or five times. Purity of plasmid: The chloroform needs to be added quickly after the ammonium acetate. An optional step to clean up the DNA from residual proteins is to add the supernatant after this step to 600 µl 1:1 phenol: chloroform.

Notes on the protocol Dissolve the pellet at 4°C for two hours to overnight. Extended storage recommended at -20°C. We normally dissolve the pellet initially in 50-100 µl TE; if after dissolving overnight there is significant undissolved DNA in the wells of an agarose gel, then more TE should be added.

Plasmids are easily visualized using standard agarose gel electrophoresis Agarose (0.7%) gel electrophoresis of plasmid extracts from seven Shigella strains, with an E. coli positive control. Note the presence of multiple plasmids in most lane. The largest band seen in most lanes is probably the 200 kb Shigella-specific virulence plasmid (Escobar-Paramo, 2003).

ULTRA PlasmidTM与同类产品的比较 1 2 3 4 5 6 7 8 1，2，5 是用本产品提取的质粒DNA 3, 4, 6, 7,8 是用其它同类产品提取的质粒DNA 其它方法可见RNA等杂质成分 1, 2, 3, 4 均为用ULTRA PlasmidTM提取的质粒DNA， 4 是用ULTRA PlasmidTM技术提取质粒后，再调节质粒超螺旋DNA，开环DNA，线状DNA组成，然后再上电泳

ULTRA PlasmidTM技术提取的质粒应用在电转染实验中 ULTRA PlasmidTM技术提取的GFP质粒电转染入肿瘤细胞内，24小时候在莹光显微镜下观察细胞表达了质粒的GFP蛋白质

可能的问题及解答 产量低： 1)质粒种类不同，质粒的拷贝数也不同。 2) 菌株的种类 3）接种的情况4）抗菌素加入的 量 5）培养液的种类 6）质粒本身含碱基个数也影响质粒的数量 质量差加入裂解溶液后，不要高速振荡，更不要用任何电动器来振荡。轻柔地颠倒混匀。 第一步离心后，抽取上清液。不要试图抽尽所有的上清液，这样会碰沉淀物（就是杂质部分）。 RNA污染：可加入0.5μl RNaseA ，37℃30分钟。 基因组DNA污染：复性时间太长

The end

DNA Analysis Downstream techniques can: Reveal how organisms are related Identify cryptic species Locate mutations in DNA http://www.biocompare.com/itemdetails.asp?itemid=687320 http://www.cdc.gov/hemochromatosis/training/images/dna.jpg

What does DNA look like? Double Helix Like a Twisted Ladder What is DNA? What does DNA look like? Double Helix Like a Twisted Ladder

What does DNA look like? What is DNA? Sugar Phosphate Backbone (Sides of Ladder) Nitrogenous Base (Rungs of Ladder)

The DNA ladder is made up of building blocks called nucleotides. What is DNA? The DNA ladder is made up of building blocks called nucleotides. What is a nucleotide? Adenine Cytosine Guanine Thymine Phosphate Group Base Deoxyribose sugar

The 4 Bases A Adenine C Cytosine T Thymine G Guanine

The 4 Bases A C T G

Evidence Collection Evidence Storage: Best bet is dry and cold. This reduces rate of bacterial growth and DNA degradation.

Organic Extraction Concentration of DNA If a small amount of DNA is targeted, the resulting solution will be dilute. One method is precipitation by ethanol. Precipitate centrifuged; supernatent removed.

Organic Extraction Concentration of DNA

轻柔地颠倒混匀10次左右，溶液逐渐变得清亮，注意不要用振荡器振荡 离心10,000-12,000rpm 5分钟，取上清液放入新管中，加入0.７ml 的异丙醇（用零下20度以下储存着的异丙醇，室温的异丙醇也可，产量稍低于冷冻的异丙醇），颠倒混匀,10,000-12,000rpm10分钟，沉淀物用75%乙醇洗1-2次，加入75%乙醇后颠倒混匀，倒掉乙醇。 若需要更纯净的质粒，在步骤4沉淀后，可加水100ul溶解质粒，再加350ul 的异丙醇，10,000rpm 10 ，沉淀物用75%乙醇洗1-2次，

For large (30 – 200 kb) and low copy plasmids Although some plasmids are small (e.g. 2 – 10 kilobases) and replicate solely in tandem with their host cells, many plasmids encode their own machinery for mobilization and self-transmission to other host cells. Such plasmids – referred to as “mobilizable” or “self-transmissible”, depending upon the functions they encode – are typically large (e.g. 30 – 200 kb) and occur in low copy numbers in the cell.

Sudden appearance of “white” precipitate after centrifugation; possible bi-products of 20 % SDS Crystallized insoluble white precipitate located on bottom of ependorf tube; signs of contaminants