Idd Ramathani , IITA
Fen Beed Idd Ramathani , IITA-Uganda Introduction DNA Extraction is the isolation and purification of DNA (deoxyribonucleic acid) DNA extraction is used to isolate… Mitochondrial DNA Genomic DNA DNA can be extracted from… • Cells or tissues • Environmental samples DNA Extraction is not used to… Isolate proteins Give information about gene expression Idd Ramathani IITA-Uganda -plant pathology After DNA is extracted, it is used as a template in further molecular techniques such as… PCR (polymerase chain reaction) RFLP (restriction fragment length polymorphism) Southern Blotting Conventional methods of DNA extraction DNA Extraction Using the Sap Extractor (based on method of Clarke et al., 1989). Genomic DNA Isolation (based on method of Saghai‐Maroof et al., 1984)* Materials needed 1. CTAB buffer at 65OC 2. Microfuge tubes (double sets) 3. Mortar and Pestle 4. Liquid Nitrogen 5. Microfuge 6. Absolute Ethanol (ice cold) 7. 70 % Ethanol (ice cold) 8. 7.5 M Ammonium Acetate 9. 55o C water bath 10. Chloroform : Iso Amyl Alcohol (24:1) 11. Water (sterile) 12. Agarose 13. 6x Loading Buffer 14. 1x TBE solution 15. Agarose gel electrophoresis system 16. Ethidium Bromide solution Idd Ramathani IITA-Uganda -plant pathology DNA extraction from plant tissues –CTAB modification 1. Grind 100 mg leaf tissue in 2 ml of extraction buffer. (use 50 mg in case of dehydrated sample) 2. Pour sap into a new and sterilized microfuge tube 3. Add 33 μl of 20% SDS 4. Vortex briefly and incubate in a water bath at 65 oC for 10 minutes 5. Allow to cool to room temperature and add 160 μl of 5 M Potassium acetate 6. Vortex and centrifuge at 10,000 g for 10 minutes 7. Remove the supernatant (about 400 μl) into another eppendorf tube 8. . Add 200 μl of cold iso‐propanol 9. Mix gently and keep on ice or at 4 oC for 15 to 20 minutes 10.Centrifuge at 10,000 g for 10 minutes to sediment the DNA 11. Decant the supernatant gently and ensure that the pellet is not disturbed 12. Add 500 μl of 70% (v/v) ethanol to the pellet (this is to wash it) and centrifuge at 10,000 g for 5 to 10 minutes 13. Decant the ethanol and air dry the DNA (at 37 oC) until no trace of alcohol can be seen in the tube. 14.Dissolve DNA pellet in 50‐100 μl of TE buffer 15. For PCR, dilute stock DNA to 1:50 or 1:100 (v/v) in TE buffer and use 1 or 2 μl per 12.5 μl reaction PCR Conventional DNA extraction method Plant Genomic DNA Extraction using CTAB based on method of Saghai‐Maroof et al., 19841)* Minimum of 10 steps to be followed. Time duration •Incubation – 60-90 minutes Modification protocol for CTAB for BBTV extraction) . Incubation – 10 minutes •Cooling samples -10minutes Several centrifuging – 30 minutes ( 10/ 10/10 three times •Centrifuging - 10 min. Placing on ice – 20 minutes •incubate for 30 min (RNase A added • 2nd Centrifuging - 15 min Time taken to obtain DNA for a single sample 4-5 Hours without power failure and easy access to all equipment (This time doesn't include PCR preparation and running time . So the entire process from extraction to Gel electrophoresis may take a whole day or more Idd Ramathani IITA-Uganda -plant pathology The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however the fundamentals of DNA extraction remains the same. DNA must be purified from cellular material in a manner that prevents degradation. Because of this, even crude extraction procedures can still be adopted to prepare a sufficient amount of DNA to allow for multiple end uses. Such as in DNA analysis: Downstream techniques can: •Reveal how organisms are related •Identify cryptic species •Locate mutations in DNA Idd Ramathani IITA-Uganda -plant pathology Some modern Methods used for capturing DNA FTA Whatman Cards b) Phytopass c) 2 minute nucleic acid extraction Dip stick a) Advantages for using these methods Require very few chemicals from treatment to PCR preparation Samples are stored at room temperature Maintain the integrity of DNA for a very long time ( 5 years and more) Avoids transfer of pathogens across borders Easier to transport from field to any lab without any restrictions Cheap in the long run Doesn't require a lot of skills to collect Time duration is limited /short Idd Ramathani IITA-Uganda -plant pathology FTA cards treatment protocols Two methods a) using purification reagent b) using GEB and GES buffer (Direct sample extraction) Using GEB and GES buffer Step 1: Punch 0.5 cm FTA card sample (make sure to take punch from area where samples are spotted) and soak it in 500 μl of GEB buffer in a microfuge tube for about 30 min at room temperature. Use this extract immediately or store the tubes at ‐20ºC for subsequent use. Step 2: Take 5 μl of the extract from step 1 (stored extracts should be thawed and mix well) and mix with 25 μl of GES buffer. Vortex it and heat denature at 95ºC for 10 min (in a water bath). Then place tubes on ice for 5 min. Use 2‐4 μl of this preparation as template in PCR or RT‐PCR reactions. Idd Ramathani IITA-Uganda -plant pathology 2 1 Apply specimen and allow to dry completely 4 Area on the FTA card 3 FTA Purification Reagent Washes Place the disk in a PCR tube and wash three times with FTA Purification Reagent. Discard used reagent after each wash. TE-1 Rinses Wash twice with TE-1 buffer and discard used buffer after each wash. Dry disk in PCR tube. 5 Idd Ramathani IITA-Uganda -plant pathology Direct to PCR Add PCR master mix directly to the disk and amplify. The Phytopass system kit Idd Ramathani IITA-Uganda -plant pathology The system allows 1. Field sampling Fast, easy and no need of additional tools No risk of cross contamination No risk of sample confusion 2. Transport and storage of samples Stable at room temperature for several weeks Easily dispatches by regular post mail 3. Preparation of crude extract Extract is ready in less than 5 minutes At least 4‐6x faster than classic protocol No freezing or grinding Idd Ramathani IITA-Uganda -plant pathology Phytopass protocol Take the Phytopass with the sampling membrane upwards. Open the Phytopass by pushing out the sampling strip up to the first stop. Place the tip of the sampling strip on a clean surface and press to separate it from the case. Pick up the sampling membrane with tweezers by the non‐ covered area. Put the membrane in a 10ml tube with a cap containing 1.5ml KAJI buffer stored at 40C. Vortex the tube for 30 seconds or vigorously hand agitate it until some crystals lay in the bottom of the tube. Incubate for 2minutes at less than 40C. Pipette 500µl of the supernatant crude extract. This extract can be stored for 3‐4 months at ‐200C. Avoid melting/freezing phases that rapidly degrade the extract. Preferably aliquot the crude extract in small volumes before storage. Idd Ramathani IITA-Uganda -plant pathology Illustration‐ sampling to PCR Idd Ramathani IITA-Uganda -plant pathology 2 MINUTE NUCLEIC ACID EXTRACTION DIPSTICK: These kits have been developed by CSL and are based on the (serological) Lateral Flow Device for plant pest identification. Basically part of the LFD membrane is placed in the PCR tube and PCR is direct from here Idd Ramathani IITA-Uganda -plant pathology Procedure with 2 minute nucleic acid extraction dipstick Disrupt sample by placing <0.5g or 4cm2 of leaf material into the bottle provided. Replace the lid and shake the bottle with the sample for between 15s seconds and 2 minutes1. It is necessary to break down the tissue, but not completely homogenise it; ‘soft’ tissue needs less shaking than ‘hard’ tissue. For a new sample type it is strongly recommended that the length of shaking required be optimized in a pilot experiment before testing large numbers of samples. Transfer 100l of sample to a tube and insert the glassfibre release pad into the sample (a). Allow the dipstick to run for 2 minutes. Cut a section of membrane (1x2mm) from centre of device (b). Place membrane directly into PCR master mix (c) – cycle as usual. Idd Ramathani IITA-Uganda -plant pathology Idd Ramathani IITA-Uganda -plant pathology INTERPRETATION OF RESULTS Idd Ramathani IITA-Uganda -plant pathology
© Copyright 2024