1. What is DNA purification? Lyse cells or tissues Inactive endogenous nucleases Separate DNA from other cellular components – Proteins – Lipids – RNA – Others

2. What samples can be used? Tissue Whole blood Buffy coat Saliva Buccal cells/swabs Urine Others (bones, hair, nails…) +++ + DNA

3. Collect whole blood Collect blood in EDTA vacutainer tube Process within 24 h, keep at RT or 4°C Store up to 5 days at 4°C Long term storage at -80°C [but caveats!] DNA degradation occurs with time Excellent DNA quality and quantity Suitable for sequencing and genotyping Additional information can be obtained – hormone levels, etc.

4. DNA is in the “buffy coat” (WBCs) Centrifuge 2000 X g for 15 min at RT Remove plasma Collect buffy coat in 1.5 ml tube Store at 4°C (days) or -80°C (years) [Not Frost-free!] Plasma: 55% of total blood Buffy coat: Leukocytes and platelets <1% of total blood Erythrocytes: 45% of total blood

5. Collect whole blood-FTA elute cards Buy from Fisher Scientific (Whatman) Apply 12 to 40 ul whole blood FTA Elute Matrix is chemically treated with reagents: – lyse cells – denature proteins – expose nucleic acids Electron micrograph showing DNA entrapped with the FTA matrix.

6. FTA Elute Cards DNA is stable for years at RT DNA is recovered through elution with water and heat from a TINY paper-punch Shipped in an envelop to our lab DNA yield is low Good for PCR

7. Oragen.DNA/saliva kits (DNA Genotek) May take 2 to 5 minutes to obtain saliva sample Painless, non-invasive collection Stable at room temperature for years High DNA quality and quantity DNA is equivalent to DNA obtained from blood Good for PCR, Illumina GoldenGate assays, OpenArray…

8. Saliva-Mouthwash Dispense 10 ml of Scope mouthwash into a 50 ml tube Collect buccal cells by vigorously swishing for ≥30 sec Spit mouthwash back into tube Keep at room temperature (up to months) Good quality DNA and quantity Suitable for PCR, Illumina GoldenGate Assays…

9. How to choose a DNA purification technique? Downstream applications (PCR, GoldenGate assays…) DNA bank for other studies Commercial kits vs in house versions – Qiagen kits: Reliable Good DNA quality Yield vary according to kit – 96 well plate – Miniprep – Maxiprep DNA Quality DNA Quantity $$$

10. Differences among collection techniques Collection methodPriceDNA quantity* EasePCRGoldenGate SNP assay Next Gen Libraries PFGE Whole blood $$++/++++√√√~ Buffy coat $$++++√√√√ FTA Elute Cards $+++√ --- Mouthwash $+++++√√~- Oragen.DNA/saliva $$$+++ √√√- * ++ or +++ allows DNA banking for further testing Lysis Buffer$+++ √√√- RNA Later$$+++++√√√N/A Citrate Buffer$+++ √√√√ Liquid N2 or -80$$$+++/++√√√√/~

11. DNA Quality Matters May not be enough for a library