1. Chelex ® Extraction

2. Learning Objectives Competence in extraction of different biological stains. Knowledge of the theory of DNA Isolation using Chelex ® Extraction methods Knowledge of advantages and disadvantages of Chelex ® Overview of 5% Chelex ® extraction procedures

3. Overview of DNA Process DNA EXTRACTION DNA QUANTITATION DNA AMPLIFICATION CAPILLARY ELECTROPHORESIS

4. DNA Facts A WBC or epithelial cell (diploid cell) contains approximately 6 pg of DNA –Blood ~1.5  g/drop –Saliva ~50-500 ng/drop –Hair (plucked) ~ 300 ng Sperm cells (haploid cell) contains approximately 3 pg of DNA –Semen ~ 10  g/drop

5. Clean Techniques and Other Considerations Wear gloves Use a barrier to open tubes (tube opener or Kimwipe) Use only sterile filtered pipette tips Use sterile deionized water Run SOP specified controls (reagent blank, etc) Make Chelex ® per laboratory SOPs

6. Chelex ® Extraction Chelex ® 100 is an ion exchange resin that is added as a 5% solution (wt/vol). Chelex ® is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups and bind metal ions such as magnesium (Mg 2+ ). By removing the Mg 2+ from the reaction, nucleases are inactivated and the DNA is protected.

7. Preparing for Extraction Things you may need to prepare in advance: –sterile 1.5 ml microcentrifuge tubes –sterile deionized (DI) water –sterile TE Buffer –sterile spin baskets or separator cups –set incubator to 56ºC –set water bath to 100ºC

8. Preparing a 5% Chelex ® Suspension Add 0.5 g Chelex ® 100 (100-200 mesh, sodium form) to a sterile beaker or suitable glass container Add10 ml sterile DI water Mix in a container on a hotplate with a stir bar inside. Check pH. –Values should be approximately 9 to 11. Adjust pH with a solution of NaOH as needed. –Make fresh daily. Keep mixing on a stir plate while pipetting with a wide bore pipette tip.

9. Chelex ® Extraction Process Many protocols include an initial wash step. Generally this is done by adding ~ 1 ml of sterile DI water to the sample. –This aids in the removal heme and other proteins that inhibit PCR. –This can be done at room temperature or 37ºC. –The incubation times may vary. –After the incubation the sample is centrifuged and the supernatant is removed and discarded. NOTE: leave approximately 30-50  l of supernatant. A 5 % Chelex ® suspension is added to the tube and incubated at ~ 56 o C –Incubation times can vary from 30 minutes to overnight depending upon the sample type –This step is used to lyse cells. –Chelex ® binds the Mg 2 + ions which inactivates nucleases.

10. Chelex ® Extraction Process, cont. Some procedures call for the addition of Proteinase K at this step. Proteinase K functions to break down proteins, specifically histones which are responsible for packaging DNA. The sample is vortexed and then boiled at 100 o C for ~ 8 minutes. This causes cell membranes to rupture, destroys cellular proteins, and denatures the DNA to yield single-stranded (SS) DNA.

11. Chelex ® Extraction Process, cont. After the boiling step, some procedures include a vortexing step. The tube containing the sample and Chelex ® suspension is then centrifuged, the beads and cellular debris are pelleted, leaving the supernatant containing DNA that is used for quantitation and PCR.

12. Overview of a Chelex ® Extraction ~1ml sterile deionized water sample Incubate 15 - 30 min at 37°C Vortex Centrifuge Remove supernatant (except 50-30  l) ~100-200  l Chelex ® 56°C for 30 min to overnight Vortex Centrifuge Store Microcon (if needed) 100°C 8 minutes

13. Centrifugal Filter Units Many laboratory procedures include a purification and concentration step using a centrifugal filter unit such as the Microcon ® 100 or Centricon ® –Excellent recovery of DNA samples with recoveries typically > 95%. –Used to concentrate, desalt, and purify proteins, antibodies and nucleic acids –Ideal for low level/dilute DNA solutions –Allows products less than 100,000 Daltons to pass through, therefore retaining DNA in the filter

14. Purification and Concentration –Initial step Assemble unit by inserting filter into filtrate/collection tube. Add sample and buffer. Centrifuge at low speed. Remove the filter unit from the filtrate tube and discard the filtrate. –Wash step Re-Insert the filter unit into the filtrate tube and add buffer to the sample reservoir and cap. Centrifuge at low speed and discard the filtrate and filtrate tube. –Recovery Step Add the appropriate amount of buffer to the filter. Invert and transfer the filter into a new retentate tube. Centrifuge at low speed. DNA is in the retentate tube.

15. Storage of DNA DNA can be stored refrigerated (4 o C) for short term storage. DNA should be stored frozen (approximately -20 o C ) for long term storage. Avoid repetitive freeze thawing of DNA, since this can cause degradation.

16. Chelex ® Extraction Considerations The Chelex ® extraction process denatures double stranded DNA and yields single stranded DNA. Care should be taken not to transfer any Chelex ® beads to PCR reaction as this can inhibit PCR Some studies show low extraction efficiency on low level and compromised samples--organic or other suitable extraction methods may be a better choice for some sample types

17. Chelex® Extraction-Advantages & Disadvantages Advantages: –Quick –Single tube reaction –Non-toxic –Cost effective Disadvantages: –Ineffective in removing some inhibitors –Yields single stranded DNA (may be problematic with some downstream methods) –May have low yield for compromised and/or low level samples