IMBB Genomic DNA purifica8on

Transcription

1 IMBB 2013 Genomic DNA purifica8on

2 Why purify DNA? The purpose of DNA purifica8on from the cell/8ssue is to ensure it performs well in subsequent downstream applica8ons, e.g. Polymerase Chain Reac8on (PCR), microsatellite analysis etc. Ideally, the DNA should be free of contamina8on with Protein Carbohydrate Lipids Other nucleic acid (i.e. DNA free of RNA) Tannins, phenolics

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4 Genomic DNA extrac8on from animal 8ssue Silica spin column purifica8on of DNA Prepare lysate using Diges8on Buffer Apply lysate to column and spin Apply wash buffer 1 to column and spin Apply wash buffer 2 to column and spin Elute DNA with low salt buffer

5 Genomic DNA extrac8on from animal 8ssue Chelex Method Add 8ssue sample to 20% (w/ v) Chelex in Water Heat 95 o C for 5 min Centrifuge Remove supernatant

6 Genomic DNA extrac8on from plant leaves: Modified Dellaporta method Lysis in SDS- DTT extrac8on buffer Precipitate proteins Breaks open cells and releases DNA Forms complexes with lipids and proteins, causing them to precipitate out of solu?on Chloroform extrac8on RNase treatment Chloroform extrac8on Isopropanol precipita8on Ethanol precipita8on Dry DNA pellet Redissolve Digests RNA Purifies and concentrates the DNA

7 Polymerase chain reac8on

8 What is PCR? The polymerase chain reac8on (PCR) is a rela8vely simple technique developed in early 1980 s to make many copies of sequence- specific DNA fragments in vitro. Also called DNA amplifica8on. PCR is one of the most useful techniques in biosciences labs today due to its speed and sensi8vity. Tradi8onal techniques to amplify DNA require days or weeks; PCR can be performed in as li`le as 2-3 hours. Many molecular analyses require the input of significant amounts of biological material; PCR requires as li`le as one DNA molecule. These features make PCR extremely useful in basic research and commercial applica8ons: DNA (and RNA) cloning DNA (and RNA) detec8on (e.g. diagnos8cs) DNA (and RNA) quan8ta8on Genotyping DNA- based iden8fica8on (DNA Barcoding)

9 What is PCR? The polymerase chain reac8on (PCR) is a rela8vely simple in vitro technique to amplify (make mul8ple 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 What is PCR? The polymerase chain reac8on (PCR) is a rela8vely simple in vitro technique to amplify (make mul8ple 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)

11 How does PCR work? The method involves using a pair of short DNA sequences called primers, or oligonucleo8des, which are made in the laboratory. The primers are designed to be complimentary to the segment of the DNA to be amplified. The reac2on A sample of target DNA is mixed with the primers 4 nucleo8des (dntps) (the building blocks of DNA), a DNA polymerase (DNA replica8on enzyme which synthesises new copies of DNA) Reac8on buffer

12 PCR Basics Step 1 The reac8on is heated to about 95 o C to denature the DNA (strand separa8on). This is called denatura8on.

13 PCR Basics Step 1 The reac8on is heated to about 95 o C to denature the DNA (strand separa8on). This is called denatura8on.

14 PCR Basics Step 2 By reducing the reac8on temperature to about o C, the primers in the reac8on specifically bind ( anneal ) to complementary regions on the target DNA. This is called primer annealing or annealing.

15 PCR Basics Step 3 The reac8on 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 elonga8on 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).

16 PCR Basics The three steps, or cycle, is repeated mes. As PCR progresses, the DNA generated is itself used as a template for replica8on, sehng in mo8on a chain reac8on in which the DNA template is exponen8ally amplified. (The amount of target DNA is doubled with each cycle.)

17 A PCR includes Buffer with magnesium Reac8on tube DNA from sample Target DNA (template) Taq DNA polymerase Primer 1 Primer 2 Deoxyonucleo8de triphosphates (dntps)

18 Aier mixing these components, the reac8on tube is placed into a thermocycler, which takes the reac8on through a series of three different temperature steps for varying short amounts of 8me (30-60 sec). This temperature series is referred to as one cycle of amplifica8on. Each cycle consists of the following 3 steps:

19 One PCR cycle A typical PCR has cycles

20 PCR movie PCR movie for IMBB_2013.flv

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

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

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

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

25 Thank you