From the Seed Sample to DNA II: DNA Isolation, Quantification, &

Normalization

Beni Kaufman

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

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 OVERALLTESTING SUCCESS

DEPENDS ON DNA ISOLATION

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

Isolating DNA…

Grinding:mechanical breakdown

of the cell wall

Dissolving membranes

Pulling out DNA, or impurities

Cleaning…

Grinding

There is no “off the shelf” grinder suitable for AP

testing…

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)

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.

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

Organic Extraction• Phenol/Chloroform

– denatures and extracts proteins

• Ethanol/high salt – Differential precipitation of DNA

Lengthy

Labor intensive

Automation hostile

Safety Issues

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

Magnetic Clearing

• Coated paramagnetic beads– Silica– proprietary– Increased binding

kinetics/efficiency– Enhanced removal of

contaminants– Commercially available

ChargeSwitch, Wizard, MagAttract

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…

Quantification

• Spectrophotometer– Absorbance at 260 nm

• (UV illuminator)• Fluorometer

– Hoechst Dye– PicoGreen

Spectrophotometry• Concentration = OD260 * 50 g/ml

(dsDNA) * dilution factor• Purity

– Measure of proteinsOD260:OD280 = 1.8

– Measure of phenolics/chaotropic salts

OD260:OD230 > 1.5

– Measure of particulatesOD330

• Simple & non-destructive, • Narrow range 5 g/ml to 90

g/ml, easy to over estimate due to contaminates (RNA, ssDNA, nucleotides, phenols, proteins)

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)

Input raw fluorescence values for standards.

Input standard curve information.

Scroll down for the standard curve.

Input raw fluorescence values for unknowns.

Concentrations given here.

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

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

Volume Initial, Concentration Final, Concentration Initial

Throughput/Automation

• At isolation, quantification, and normalization