Presentation on theme: "DNA Extraction Outline Purpose of DNA extraction"— Presentation transcript:
1 DNA Extraction Outline Purpose of DNA extraction Review the main steps in the DNA extraction protocol and the chemistry involved in each stepImage of DNA
2 Purpose of DNA Extraction To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc
3 Basic Protocol Most DNA extraction protocols consist of two parts A technique to lyse the cells gently and solubilize the DNAEnzymatic or chemical methods to remove contaminating proteins, RNA, or macromoleculesIn plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell.LysisPrecipitationWashResuspension
4 A comparison of DNA extraction methods used in research labs as opposed to classroom labs Lysis: grind in Liquid N2 and use detergentPrecipitation Part I: phenol/chloroform extraction to get rid of proteinsPrecipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNAPrecipitation Part III: addition of ethanol to pull DNA out of solutionWash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.ClassroomLysis: grind in mortar/pestel and use detergentPrecipitation Part I: NONE (chemical are too dangerous!)Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNAPrecipitation Part III: addition of ethanol to pull DNA out of solutionWash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.
5 LYSIS: In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes)The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves)Then the addition of a detergent in the which breaks down the cell membranesDetergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranesThe end result of LYSIS is that the contents of the plant cells are distributed in solution.
6 PRECIPITATION (In a research lab): This a series of steps where DNA is separated from the rest of the cellular componentsIn a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNAPhenol denatures proteins and dissolves denatured proteins.Chloroform is also a protein denaturantTHIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!The second part of research lab DNA precipitation is the addition of saltsThe salts interrupt the hydrogen bonds between the water and DNA molecules.The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanolIn the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.The DNA is pelleted by spinning with a centrifuge and the supernatant removed
7 PRECIPITATION (In a classroom lab): This a series of steps where DNA is separated from the rest of the cellular componentsIn a classroom lab, DNA precipitation involves the addition of saltsThe salts interrupt the hydrogen bonds between the water and DNA molecules.The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanolIn the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.The DNA is pelleted by spinning with a centrifuge and the supernatant removedNote: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab!
8 Washing and Resuspension: The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA.Resuspension:The clean DNA is now resuspended in a buffer to ensure stability and long term storage.The most commonly used buffer for resuspension is called 1xTE
9 Overview of DNA Extraction Break down the cell wall and membranesCentrifuge to separate the solids from the dissolved DNAPrecipitate the DNA using isopropanolCentrifuge to separate the DNA from the dissolved salts and sugarsFlowchart of the various stepsDissolve DNAWash the DNA pellet with Ethanol and dry the pellet
10 Checking the Quality of your DNA The product of your DNA extraction will be used in subsequent experimentsPoor quality DNA will not perform well in PCRYou will want to assess the quality of your DNA extraction using the following simple protocol:Mix 10 µL of DNA with 10 µL of loading bufferLoad this mixture into a 1% agarose gelAnalyze results (the following slides provide guidance)
11 Expected Results in a Research Lab Below is an agarose gel that has 5 genomic DNA samples from various plants.Note that the DNA runs at a very high molecular weight and as a clear, thick band.This DNA was extracted in a research lab under optimal conditionsImage of Matt's perfect extraction1 kbp and 100 bp laddersGenomic DNA of 5 species of cereals
12 Analyzing DNA Samples in a Research Lab If properly done, genomic extraction should result in bright bands in the very high base pair range of a gel electrophoresis.Sizes of Genomic DNA for various Species in kbpE. Coli ,640,000bpYeast 12,100,000bpFruit Fly 140,000,000bpHuman 3,000,000,000bpPea 4,800,000,000bpWheat 17,000,000,000bpThe genomic fragments run at ~12kbp because they are sheared during extraction
13 Expected Results in a Classroom Lab A B C D EUsing the protocol in the Cereal Genomics module, the genomic DNA extracted will look different than the optimized DNA extraction on the previous slide (this is mainly due to the missing phenol/chloroform step)This is expected. Even though this genomic DNA preparation is not perfect, it is suitable for use as a PCR templateLane A: Barley Lane B: CornLane C: OatLane D: RiceLane E: WheatLadderPut on the pictureNote that the DNA has sheared (particularly for wheat) – broken up into numerous fragments and is not a clean single band at the top – these are the mid-ranged sized fragments ( ,000bp size range)The bright bands at the bp range are RNA, which also gets extracted using this protocol
14 Analyzing DNA samples in a Classroom Lab A B C D EAnalysis of samples:Barley (A): This sample is fineCorn (B): This sample is fineOat (C) : This sample is fineRice (D) : This sample is fineWheat (E): This sample has severedegradation, can work for PCRbut should re-extractLadder