BIO 430: Forensic DNA Analysis

1. BIO 430:Forensic DNA Analysis Lecture 5 � DNA Extraction

2. DNA Extraction After Biological samples are collected/ stored, DNA must be separated from other cellular material ? Why?

3. DNA Extraction Overall steps: Lyse cells Lyse nucleus DNA separated/protected from nucleases, heme etc.

4. DNA extraction Three primary techniques: Organic extraction Chelex extraction FTA paper

5. Organic Extraction May be used for either RFLP or PCR Removes proteins and other cellular components from DNA/RNA Yields relatively purified dsDNA preparations

6. Organic Extraction 1 � Phenol/Chloroform

7. Organic Extraction 2 � Ethanol Precipitation To DNA containing aqueous phase, add sodium acetate (salt) and 100% cold EtOH Mix well; incubate at -20�C for ~20 min Centrifuge at max speed for 10-15 min Pour off supernatant; add 70% EtOH (to wash pellet) and centrifuge Pour off EtOH; allow DNA pellet to dry Resuspend in appropriate volume of TE or H2O

8. Organic Extraction Pros: yields relatively pure, high molecular weight DNA DNA is double stranded � good for RFLP Cons: Time consuming Requires sample to be transferred to multiple tubes � increases risk of contamination Involves use of hazardous (and smelly!) chemicals

9. Chelex Extraction Chelex� is a chelating resin that has a high affinity for polyvalent metal ions Chelex� beads bind to metal ions - e.g. Magnesium Removing magnesium from solution inhibits nucleases Prevents DNA degradation Results in denatured (single stranded) sample DNA

10. Chelex extraction Put sample in tube Add 5% Chelex� beads; vortex Boil at 100�C (why?)

11. Chelex extraction Pros: Relatively fast Can extract directly from cloth (stain) Minimizes contamination � uses only a single tube Removes PCR inhibitors Con: Results in single-stranded DNA � not useful for RFLP

12. FTA� Paper FTA� originally developed by Lee Burgoyne for long-term storage of DNA Contains four chemicals that protect DNA from nucleases and bacteria Can be used directly to extract DNA for analysis

13. FTA� Paper extraction Spot blood/saliva on paper Dry for 30 min Cells lyse on contact with paper DNA immobilized to paper Punch a small hole from paper Place punch in tube; wash Use washed paper punch directly in PCR reaction

14. FTA� Paper - punchers Hand-held punchers Automated Punchers

15. FTA� Paper Pros: Very quick Useful for both storage and extraction Cons: Not useful for RFLP Paper punched �jump� because of static electricity � potential contamination

16. Solid-Phase Extraction DNA from lysed cells binds to solid material Impurities are washed away Examples: Qiagen � Silica-based Dynal - Magnetic

17. Silica-based extraction

18. Silica-Based Extraction Pro: Quick Highly purified DNA Con: Multiple sample transfer Increase risk of contamination

19. Magnetic Beads Magnetic beads are coated with DNA antibodies to bind to DNA:

20. Magnetic Beads Automated version:

21. Magnetic Beads Pro: Very fast, may be automated Highly purified DNA Excellent for liquid blood Con: Cannot be used directly on stain i.e. need to remove cells from stain substrate (cloth, etc.) Very expensive

22. Differential DNA Extraction Separates epithelial (female) and sperm (male) cells from vaginal, rectal, oral and penile swabs in sexual assault cases Concept: Epithelial cells are easier to lyse than sperm cells

23. Differential Extraction Add digest buffer (SDS, EDTA, Proteinase K) to Epithelial/sperm cell suspension Only epithelial cells are lysed Incubate cells at 37�C Centrifuge; Separate Pellet and Supernatant Pellet: intact sperm Supernatant: epithelial cell lysate (with DNA)

24. Differential Extraction To pellet (sperm) add Lysis buffer + DTT (dithiothreitol) - Why? perform standard DNA extraction techniques on lysed epithelial cell fraction and lysed sperm cell fraction

25. Differential DNA extraction