IMBB Genomic DNA extrac7on

Transcription

1 IMBB 2014 Genomic DNA extrac7on

2 Why extract purify DNA? The purpose of DNA purifica7on from the cell/7ssue is to ensure it performs well in subsequent downstream applica7ons, e.g. Polymerase Chain Reac7on (PCR), microsatellite analysis etc. Ideally, the DNA should be free of contamina7on with Protein Carbohydrate Lipids Other nucleic acid (i.e. DNA free of RNA) Tannins, phenolics

3 Basic procedure of DNA extrac3on uses a combina7on of physical and chemical methods. 1. Breaking open the cells open (cell lysis) - Chemical/physical methods 2. Removing membrane lipids by adding a detergent (also lyses the cells) 3. Digest proteins by adding a protease (op7onal) 4. Digest RNA by adding an RNAase DNA purifica7on from detergents, proteins, digested RNA, salts and reagents used during cell lysis step. The most commonly used procedures are: Ethanol or isopropanol precipita7on Phenol chloroform extrac7on and precipita7on Minicolumn (kit) purifica7on adsorp7on to a solid phase hyp://en.wikipedia.org/wiki/dna_extrac7on

4 Genomic DNA extrac7on from plant leaves: Modified Dellaporta method Lysis in SDS- DTT extrac7on buffer Precipitate proteins Breaks open cells and releases DNA Forms complexes with lipids and proteins, causing them to precipitate out of solu?on Chloroform extrac7on RNase treatment Chloroform extrac7on Isopropanol precipita7on Ethanol precipita7on Dry DNA pellet Redissolve Digests RNA Purifies and concentrates the DNA

5 Silica spin column purifica7on of DNA Prepare lysate using Diges7on Buffer Apply lysate to column and spin Apply wash buffer to column and spin Apply wash buffer to column and spin Elute DNA with low salt buffer

6 Polymerase chain reac7on

7 What is PCR? The polymerase chain reac7on (PCR) is a rela7vely simple technique developed in early 1980 s to make many copies of sequence- specific DNA fragments in vitro. Also called DNA amplifica7on. PCR is one of the most useful techniques in biosciences labs today due to its speed and sensi7vity. Tradi7onal techniques to amplify DNA require days or weeks; PCR can be performed in as liyle as 2-3 hours. Many molecular analyses require the input of significant amounts of biological material; PCR requires as liyle as one DNA molecule. These features make PCR extremely useful in basic research and commercial applica7ons: DNA (and RNA) cloning DNA (and RNA) detec7on (e.g. diagnos7cs) DNA (and RNA) quan7ta7on Genotyping DNA- based iden7fica7on (DNA Barcoding)

8 What is PCR? The polymerase chain reac7on (PCR) is a rela7vely simple in vitro technique to amplify (make mul7ple copies of) a specific sequence (i.e. a small region or fragment) of DNA from a complex mixture of DNA. DNA from sample Target DNA (template)

9 What is PCR? The polymerase chain reac7on (PCR) is a rela7vely simple in vitro technique to amplify (make mul7ple copies of) a specific sequence (i.e. a small region or fragment) of DNA from a complex mixture of DNA. DNA from sample Target DNA (template)

10 How does PCR work? The method involves using a pair of short DNA sequences called primers, or oligonucleo7des, which are made in the laboratory. The primers are designed to be complimentary to the segment of the DNA to be amplified. The reac3on A sample of target DNA is mixed with the primers 4 nucleo7des (dntps) (the building blocks of DNA), a DNA polymerase (DNA replica7on enzyme which synthesises new copies of DNA) Reac7on buffer

11 PCR Basics Step 1 The reac7on is heated to about 95 o C to denature the DNA (strand separa7on). This is called denatura7on.

12 PCR Basics Step 1 The reac7on is heated to about 95 o C to denature the DNA (strand separa7on). This is called denatura7on.

13 PCR Basics Step 2 By reducing the reac7on temperature to about o C, the primers in the reac7on specifically bind ( anneal ) to complementary regions on the target DNA. This is called primer annealing or annealing.

14 PCR Basics Step 3 The reac7on temperature is then raised to 72 o C. At this temperature the DNA polymerase make two new strands of the target DNA, beginning at where the primers have bound. This step is known as extension or elonga7on because the polymerase extends or elongates the primer, using the complementary strand as a template. To withstand the high temperature of the PCR, a thermostable DNA polymerase is used (e.g. Taq DNA pol).

15 PCR Basics The three steps, or cycle, is repeated mes. As PCR progresses, the DNA generated is itself used as a template for replica7on, segng in mo7on a chain reac7on in which the DNA template is exponen7ally amplified. (The amount of target DNA is doubled with each cycle.)

16 A PCR includes Buffer with magnesium Reac7on tube DNA from sample Target DNA (template) Taq DNA polymerase Primer 1 Primer 2 Deoxyonucleo7de triphosphates (dntps)

17 Aher mixing these components, the reac7on tube is placed into a thermocycler, which takes the reac7on through a series of three different temperature steps for varying short amounts of 7me (30-60 sec). This temperature series is referred to as one cycle of amplifica7on. Each cycle consists of the following 3 steps:

18 One PCR cycle A typical PCR has cycles

19 PCR movie PCR movie for IMBB_2013.flv

20 Figure 8-45b Molecular Biology of the Cell ( Garland Science 2008)

21 Animal? Muscle sample DNA CO1 gene PCR CO1 gene (~1500 bp) PCR PCR product (~ bp)

22 ? Leaf sample DNA rbcl gene PCR rbcl gene (~1430 bp) PCR PCR product (~600 bp)

23 Bioneer AccuPower PCR PreMix is a ready- to- use PCR reagent, in individual PCR tubes, lyophilised and stable.

24 Thank you