1. From the Seed Sample to DNA II: DNA Isolation, Quantification, & Normalization Beni Kaufman

2. The Importance of DNA Isolation: Quality of DNA determines PCR efficiency, hence, the detectability & sensitivity of the test. PCR efficiency is affected by the: concentration of impurities: Directly; enzyme inhibitors, or Indirectly – –impurities binding the DNA and effectively making it unavailable to the enzymatic reaction DNA Isolation Quantification Normalization PCR Set-up PCR

3. Also, PCR efficiency is affected by: the integrity of DNA molecules (length of fragments, shredding, degradation) Both impurities and degradation may affect quantification – throw off reaction DNA Quantity defines representation, and the contribution to sampling error THE OVERALL TESTING SUCCESS DEPENDS ON DNA ISOLATION

4. Challenges to isolate DNA from seed: –Compared to leaf tissue: Seed contains much less DNA and much (much) more “impurities” (carbohydrates, phenols, lipids) –The most labor intensive step –The priciest step –Throughput bottle neck

5. Isolating DNA… Grinding: mechanical breakdown of the cell wall Dissolving membranes Pulling out DNA, or impurities Cleaning…

6. Grinding There is no “off the shelf” grinder suitable for AP testing…

7. Grinding Effects of particle size – …The smaller the better! –The smaller the particle size, the larger the surface area and the exposure to the extraction buffer – and therefore, the more efficient the extraction… –Representation of the lot (particles per unit mass) –Homogeneity of the mixture (particles per unit volume)

8. Dissolving Membranes Lysis by way of: –Detergents SDS CTAB –Chaotropic Salts –Alkaline Lysis –Other denaturing reagents Results in a “soup” of cellular debris and the content of the cytoplasm.

9. Pulling Out… Pulling In… Separate the soluble (DNA) from the insoluble (cell debris) by way of: –Centrifugation –Filtration Purify DNA from other solubles –Organic solvents –Columns –Magnetic clearing

10. Organic Extraction Phenol/Chloroform –denatures and extracts proteins Ethanol/high salt –Differential precipitation of DNA Lengthy Labor intensive Automation hostile Safety Issues

11. Columns Silica Column –DNA binds silica in high concentrations of chaotropic salts –Impurities are washed off –Eluted off with low ionic strength solution –Increased throughput & purity –Commercial kits NucleoSpin, DNeasy, GenElute

12. Magnetic Clearing Coated paramagnetic beads –Silica –proprietary –Increased binding kinetics/efficiency –Enhanced removal of contaminants –Commercially available ChargeSwitch, Wizard, MagAttract

13. Cleaning and Elution Alcohol wash to remove salts and other impurities. Elute or dissolve DNA in TE, water, or other low ionic strength buffer. Ready for quantification…

14. Quantification Spectrophotometer –Absorbance at 260 nm (UV illuminator) Fluorometer –Hoechst Dye –PicoGreen

15. Spectrophotometry Concentration = OD 260 * 50  g/ml (dsDNA) * dilution factor Purity –Measure of proteins OD 260 :OD 280 = 1.8 –Measure of phenolics/chaotropic salts OD 260 :OD 230 > 1.5 –Measure of particulates OD 330 Simple & non-destructive, Narrow range 5  g/ml to 90  g/ml, easy to over estimate due to contaminates (RNA, ssDNA, nucleotides, phenols, proteins)

16. Fluorometry Detection of enhanced fluorescence upon dye binding dsDNA Hoechst 33258 –Quantitate to 10ng/ml PicoGreen –Quantitate to 25pg/ml, and less sensitive to the presence of contaminants (RNA, proteins, detergents)

17. Input raw fluorescence values for standards.

18. Input standard curve information. Scroll down for the standard curve.

19. Input raw fluorescence values for unknowns. Concentrations given here.

20. Normalization Provides uniformity in testing: –To comply with the sampling scheme –To comply with validated process/assays qPCR enables in-assay normalization but logistically simple to follow uniform processing Enhances robustness of testing – higher success rate, hence, in the long run reduces cost, and on the average may improve turn around time

21. Diluent needed to add to Volume Initial to give Concentration Final. Volume Initial, Concentration Final, Concentration Initial

22. Throughput/Automation At isolation, quantification, and normalization