Extraction, Quantitation, and Evaluation of Function DNA from Various

Chapter 14 Extraction, Quantitation, and Evaluation of Function DNA from Various Sample Types Malin Ivarsson and Joyce Carlson Abstract Two vital pre-requisites for genetic epidemiology have been fullfiled during the past decade and have led to a virtual explosion of knowledge concerning disease risks. Reliable databases over genetic variation derived from, e.g. the HUGO and HapMap projects, coupled with technological advances make largescale genetic analyses and downstream bioinformatics suddenly affordable. Although recent prospective population-based biobanks have included DNA collection and purification in their planning, it is the older projects that currently are of greatest value due to the numbers of accumulated disease endpoints. In this chapter, methods to purify and use DNA derived from a variety of archival materials, including whole blood, formalin-fixed paraffin-embedded (FFPE) tissues, sera, dried blood spots (DBS), cervical cell suspensions, and mouthwash are presented and evaluated in a context of quality control guidelines to provide objective measure of the usefulness of various sample types for genetic epidemiology. Key words:  DNA extraction, Dried blood spots, Formalin-fixed paraffin-embedded tissue, Mouthwash samples, Serum, Plasma, Cervical cell suspension, Whole blood, Whole genome amplification, DNA quantification

1. Introduction Many methods have been developed for the extraction of DNA from biological substances. Early methods were often manual and time consuming, but some produced large amounts of high-quality DNA in experienced hands (1). The goal of this chapter is to present guidelines for evaluating methods, creating a basis for comparison, and selection of new methods, as more and more advanced commercial alternatives become available. The basic essential guidelines involve the evaluation of quantity, purity, structural integrity, and function. One of the basic challenges involved in biobank-related research is that we must always assume Joakim Dillner (ed.), Methods in Biobanking, Methods in Molecular Biology, vol. 675, DOI 10.1007/978-1-59745-423-0_14, © Springer Science+Business Media, LLC 2011

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that our future needs are unknown. We can, therefore, never demonstrate that a particular sample will function perfectly or that it is adequate. We can only describe its characteristics within the vocabulary of our available technology. The selection of methods for both DNA extraction and for qualitative evaluation is dictated by available sample volumes, sample DNA content, the local laboratory environment, and expected downstream applications. Our personal experience has included a total of >100,000 DNA extractions over the past decade. In general, the collection and storage of biobank samples should be performed within “Good Biobanking Practice”, including the concepts of sample identification codes, restricted access to the code key, complete tracking of samples and their derivatives, complete tracking of the chain of custody in sample handling, and reliable storage and retrieval of samples. Although our work has been done primarily on existing sample collections, some differences in yields due to collection tubes, methods or storage conditions can be noted and can guide prospective sample collection and storage. As biobank samples frequently can be suboptimal in quality and are nearly always available in limited amounts, we try to start each new project by creating a fresh, abundant, homogeneous, and representative control material typical of the sample type to be handled for method development. Pilot DNA extractions and evaluations are then performed on this material before consuming the unique biobank samples. The inclusion of one or a few such control samples in each extraction batch and calculation of yield statistics over time enable the detection of changes in quality of reagent batches and technical problems within the chosen system.

2. Materials All water is sterile and Millipore filtered and all pipette tips that are introduced into stock DNA solutions are disposable, sterile, and equipped with aerosol barriers to prevent contamination from one sample to another. 2.1. DNA Extraction from EDTA Whole Blood: Qiagen Minipreparation Protocol

1. QIAamp DNA Mini kit (Qiagen).

2.2. DNA Extraction from EDTA Whole Blood: Qiagen Autopure LS Maxipreparation Protocol

1. Autopure LS Maxi-preparation kit (Qiagen).

2. 99.9% Ethanol (Kemetyl AB).

2. 100% Isopropanol (Fisher Scientific). 3. 70% Ethanol (Kemetyl AB). 4. Proteinase K (Saveen Werner) Store at +4°C. 5. Autopure LS extraction robot (Qiagen, Hilden, Germany).

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2.3. DNA Extraction from Dried Blood Spots

1. EZNA Forensic DNA reagent kit. (Omega Bio-Tek).

2.4. DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues

1. Digestion buffer: 50 mM Tris–HCl, pH 8.3, 1 mM EDTA, 0.5% Tween 20 (Merck-Schuchardt).

2. 70% Ethanol (Kemetyl AB).

2. Paraffin beads (make). 3. QIAquick Gel Extraction Kit (Qiagen). 4. 100% Isopropanol (Fisher Scientific). 5. Proteinase K (Saveen Werner). 6. QIAamp kit (Qiagen). 7. 70% Ethanol (Kemetyl AB).

2.5. DNA Extraction from Serum and Plasma: QIAamp MinElute Virus Spin Protocol

1. QIAamp MinElute Virus Spin kit (Qiagen).

2.6. DNA Extraction from Serum and Plasma: MagNA Pure LC Total Nucleic Acid Isolation Protocol

1. MagNA Pure LC instrument (Roche Diagnostics, Penzberg, Germany).

2.7. DNA Extraction from Cervical Cell Suspensions

1. 154 mM NaCl.

2.8. DNA Extraction from Mouthwash Samples

1. Oragene DNA purification kit (DNA Genotek, Inc., Ottawa, Ontario, Canada).

2. tRNA (Sigma). 3. 70% Ethanol (Kemetyl AB).

2. Total Nucleic Acid Isolation Kit (Roche Diagnostics, Penzberg, Germany).

2. 10 mM Tris–HCl, pH 7.4.

2. 70%, 95% Ethanol (Kemetyl AB). 3. TE-buffer (konc?).

2.9. Quantitation of DNA Yield: UV Absorbance at 260 and 280 nm

1. 96-well plastic micro-titre plates for dilution (Sarstedt).

2.10. Quantitation of DNA Yield: PicoGreen Fluorescence

1. 96-well plastic micro titre plates for dilution (Sarstedt).

2. TE buffer: 10 mM Tris–HCl and 1 mM EDTA, pH 8.0. 3. Fluostar Optima (BMG, LabVision). 4. NanoDropTM (NanoDrop Technologies, Inc., Wilmington, USA).

2. 96-well black micro-titre plates for fluorescence measurements (Greiner nr 655076). 3. PicoGreen dsDNA quantitation kit (P7589 Molecular Probes, Eugene, USA). Store dark at −20°C. 4. Fluostar Optima (BMG, LabVision).

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2.11. Quantitation of DNA Yield: OliGreen Fluorescence

1. 96-well plastic micro-titre plates for dilution (Sarstedt). 2. Heating block. 3. Ice. 4. 96-well black micro-titre plates for fluorescence measurements (Greiner nr 655076). 5. OliGreen ssDNA quantitation kit (P7589 Molecular Probes, Eugene, USA). Store dark at −20°C. 6. Fluostar Optima (BMG, LabVision).

2.12. Quantitation of DNA Yield: SYBRGreen QuantiTect PCR Kit

1. SYBR-Green QuantiTect PCR Kit (Qiagen, Hilden, Germany).

2.13. Quantitation of DNA Yield: TaqMan SNP Analysis

1. TaqMan Master Mix (Applied Biosystems).

2. 7900HT sequence detection system (Applied Biosystems).

2. F2 20210G > A 40× Assay Mix (dbSNP rs1799963, Applied Biosystems). Store all reagents dark at −20°C. 3. DNA standard with genotype F2 20210 G/G of known concentration (prepared at the laboratory). 4. 7900HT sequence detection system (Applied Biosystems).

2.14. Determination of Extracted DNA Fragments: Agarose Gel Electrophoresis

1. Gel electrophoresis apparatus (e.g. MGU-502T or SGU2626T-02, C.B.S.Scientific Co., DelMar CA, USA), including tray and combs. 2. Voltage supply (e.g. EC105, E-C Apparatus Corp.). 3. UV-light board. 4. CCD camera with printer (optional for digital storage). 5. 10× TBE buffer: 0.9 M Tris, 0.9 M Boric acid and 10 mM EDTA pH 8.3. 1× working solution is prepared by dilution in water. 6. Ethidium bromide 10 mg/mL (AppliChem). Note carcinogenic! Wear gloves! Store dark at +4°C. 7. 10× Loading dye: 12.5% (w/v) Ficoll 400 and 0.0025% (w/v) bromphenol blue. Store at room temperature. Mix one part dye to nine parts sample at loading. 8. Marker XIII, 50 bp ladder (Boehering Mannheim). 9. NuSieve GTG Agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA). 10. SeaKem LE Agarose (Cambrex). 11. Erlenmeyer flask. 12. Micro-wave oven or a heating plate.

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For 0.8% gel in a 130 × 100 mm gel tray. 1.6 g NuSieve GTG Agarose. 1.6 g SeaKem LE Agarose. 75 mL 1× TBE. 3.5 mL Ethidium bromide. 2.15. Usefulness of Extracted DNA: Automated SNP Analysis

1. SEQUENOM Mass Array (SEQUENOM MassARRAY, SanDiego, USA). 2. Appropriate oligonucleotides (Metabion) with sequences as determined by the SEQUENOM software for the SNPs of interest. 3. All other reagents and equipment from the SEQUENOM Mass Array manufacturer. 4. 7900HT sequence detection system (Applied Biosystems). 5. TaqMan MGB “assay by design” 40× AssayMix (Applied Biosystems). HFE H63D Forward 5¢-GAT GAC CAG CTG TTC TGT TTG-3¢. Reverse 5¢-CCA CAT CTG GCT TGA AAT TCT ACT G-3¢. Probe 1 5¢-VIC-CGA CTC TCA TGA TCA TA-MGB-3¢. Probe 2 5¢-FAM-CGA CTC TCA TCA TCA TC-MGB-3¢. HFE C282Y Forward 5¢-GGC TGG ATA ACC TTG GCT GTA C-3¢. Reverse 5¢-TCC AGG CCT GGG TGC TC-3¢. Probe 1 5¢-VIC-CCT GGC ACG TAT AT-MGB-3¢. Probe 2 5¢-FAM-ACC TGG TAC GTA TAT C-MGB-3¢. 6. No AmpErase UNG Master mix (Applied Biosystems). 7. Control DNA with known genotypes for the both polymorphisms.

2.16. Usefulness of Extracted DNA: SNP Analysis by RFLP

1. 10× PCR buffer, GenAmp PCR buffer II (Applied Biosystems). 2. 25 mM MgCl2, GenAmp MgCl2 solution (Applied Biosystems). 3. 100 mM dNTPs, GenAmp dNTP blend (Applied Biosystems). 4. Distilled water. 5. 10 mM Oligonucleotides (DNA technology). HFE H63D Forward 5¢-GAC CTT GGT CTT TCC TTG TTT GAA GC-3¢.

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Reverse 5¢-GGG CTC CAC ACG GCG AC-3¢. HFE C282Y Forward 5¢-CCA GGG CTG GAT AAC CTT GGC T-3¢. Reverse 5¢-CCC AGA TCA CAA TGA GGG GCT G-3¢. 6. AmpliTaq Gold DNA polymerase (Applied Biosystems). 7. Control DNA with known genotypes for the both polymorphisms. 8. Restriction enzyme BclI and 10× buffer G (Fermentas). 9. Restriction enzyme RsaI and 10× buffer Tango (Fermentas). 2.17. Usefulness of Extracted DNA: Multiple Displacement Amplification

1. GenomiPhi DNA amplification kit (GE Healthcare). Store at −80°C. 2. TE buffer: 10 mM Tris–HCl and 1 mM EDTA, pH 8.0.

3. Methods 3.1. DNA Extraction from EDTA Whole Blood: Qiagen Mini-preparation Protocol

This method is used for extracting DNA from EDTA whole blood. Start with 200 mL EDTA whole blood according to the Purification of DNA from whole blood protocol in the QIAamp 96 DNA Blood Handbook (see Note 1).

3.2. DNA Extraction from EDTA Whole Blood: Qiagen Autopure LS Maxipreparation Protocol

This method is used for extracting DNA from EDTA whole blood. DNA is extracted from 4.5 mL EDTA whole blood using the Gentra Autopure LS robotic system protocols for fresh blood ( A SNP in the human genome on the Applied Biosystems 7900HT instrument in the absolute quantification mode according to the manufacturers’ instructions. Read the plate in allelic discrimination mode to validate the absence of GA or AA genotypes. Use a DNA standard of known concentration in a series of tenfold dilution from 107copies to a single copy of the DNA template to create a standard curve (see Note 17).

3.14. Size Determination of Extracted DNA Fragments: Agarose Gel Electrophoresis

Electrophoresis is performed as described (2) (see Note 18). Mix agarose and TBE in an Erlenmeyer flask. Cover and boil in a micro-wave oven or on a heating plate. Mix and cook until agarose is completely dissolved. Add approx. 0.04% Ethidium Bromide, mix and cool to 65°C. Pour into a gel tray that has been taped at both ends. Place combs and cool at room temperature for 30 min. To run, lace gel in the electrophoresis apparatus, cover with 1× TBE buffer, and gently remove comb. Mix nine parts sample with one part 10× loading dye and apply samples to wells. Apply the marker to appropriate wells. Close cover, and apply voltage. Run small gels at 90  V for 15–60  min and large gels at 190  V from 45 to 120 min. Photograph the finished gel exposed on a UV-light board, and determine fragment size by comparison to the marker bands (see Note 19).

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3.15. Usefulness of Extracted DNA: Automated SNP Analyses

Within a specific project, it is always desirable to demonstrate that the DNA extract obtained will function for the intended use. In biobank contexts, we frequently wish to store aliquots of the ­sample for future (unknown and unanticipated) uses. Thus, ambition must be tempered with practicality and economy – both financial and regarding the wisest use of limited amounts of sample. The testing situations should be relevant for the intended use. Analyse SNPs with the SEQUENOM Mass Array according to the manufacturers’ instructions. Mix 1–10 ng sample with 0.125 mL AsayMix and 12 mL MasterMix in a 25 mL reaction. Run the following PCR programme 50°C for 2  min, 95°C for 10 min, 50 cycles of 95°C for 15 min, and 60°C for 1 min. Analyse the samples in the 7900HT instrument set to allelic discrimination mode (see Note 20).

3.16. Usefulness of Extracted DNA: SNP Analysis by RFLP

Mix dNTPs and dilute to create a solution containing 1.25 mM of each dNTP. Create a 50 mL PCR reaction mix using 5 mL buffer, 8 mL 1.25 mM dNTP, 3 mL 25 mM MgCl2, 2 mL of each 10 mM primer, 1.25U Taq polymerase, and 5mL DNA sample or control. Run the following PCR programme: 95°C for 9 min, 30 cycles of 95°C for 40 s, 60°C for 30 s, 72°C for 1 min, and 72°C for 10 min, hold at 10°C. Cleave the HFE H63D PCR products using BclI in a 25 ml reaction containing 2.5 mL 10× buffer G, 0.4U enzyme and 9 mL PCR product. Incubate at 55°C over night. Cleave the HFE C282Y PCR products using RsaI in a 25  mL reaction containing 2.5 mL 10× buffer Tango, 0.4U enzyme, and 9 mL PCR product. Incubate at 37°C for 2 h. Visualise the fragments on a 2% agarose gel by electrophoresis as described above (Subheading 3.3.1). Compare the fragment lengths of the samples with the controls to determine genotypes (see Note 21).

3.17. Usefulness of Extracted DNA: Multiple Displacement Amplification

Use the GenomiPhi kit according to the manufacturers’ instructions. Dilute the Multiple Displacement Amplification (MDA) product tenfold in TE buffer. Incubate on a shaker overnight to dissolve DNA (see Note 22).

3.18. Conclusions

DNA extracted from EDTA whole blood using mini- or maxipreparations generally yields micro-gram amounts of high molecular weight DNA of a quality that is sufficient for successful downstream genotyping and MDA. DBS yield low amounts (ng)

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of DNA but of sufficient quality for successful genotyping and MDA. Storage of DBS samples at −20°C prevents DNA ­degradation and fragmentation. FFPE tissue usually yields relatively large amounts of DNA that can be successfully used for genotyping, preferably designed for short amplicons. However, the extraction is time-consuming and the DNA largely fragmented and unsuitable for MDA. Plasma and serum samples give low (ng) DNA yields. Plasma or serum DNA extracted with the QIAamp or MagNA Pure protocols can be successfully used for genotyping, whereas MagNA Pure extracts are not suitable for MDA. Cervical cell suspension extracts can be successfully used for the detection of viral DNA. DNA yield from mouthwash samples is generally less than 100 mg although some samples may yield no DNA, probably caused by incorrect sampling technique or leakage. Among the quantitative methods; UV absorbance, PicoGreen fluorescence, and real-time PCR, UV absorbance at 260/280 nm is the least expensive and provides the added value of the 260/280 ratio – a measure of purity. It requires and consumes the largest amount of sample, and seems least reliable at truly low levels. In the presence of RNA and/or DNA from other species, this method overestimates the native DNA content. The PicoGreen method is rapid, sensitive, and specific for dsDNA but involves the added cost of the PicoGreen reagent. Real-time PCR is the most sensitive of the three methods, detecting down to a single copy of DNA (for the human genome, this is about 3.5 pg). Its cost in 384-format is comparable to that of PicoGreen in 96-format, and it provides the additional information of functionality in a PCR reaction as well as the potential for species discrimination. In general, it is wise to consume as little sample as possible for high-quality results in all biobank-based research. Pilot studies using representative materials should be performed prior to all large projects, with the documentation of fragment size of extracted DNA, its quantity, purity, and function in project specific applications.

4. Notes 1. Prolonged heat incubation time prior to the evaluation of the DNA does not improve DNA yield (Table 1). A single freezethaw cycle (−20°C for ³48  h) generally increases the yield, presumably due to cell lysis, however, the variation in yield also increases drastically (Table  1). 200  mL frozen (−20°C) whole blood samples can yield up to 100 mg high molecular weight DNA, but only about 1.5% of the samples have yields of >20 mg DNA when determined by PicoGreen fluorescence. 2. Fresh blood yields a mean of about 150  mg DNA, blood ­frozen on the day of collection at −80°C yields a mean of

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Table 1 DNA yield from 200 mL whole blood according to length of heat incubation using the QIAamp whole blood protocol Qiagen 10 min

Qiagen 30 min

Qiagen 60 min

Qiagen frozen

N

96

96

96

96

DNA yield

pg/cell

pg/cell

pg/cell

pg/cell

Mean

  4.96

4.58

1.88

  5.99

Median

  4.84

4.64

1.67

  4.76

S.D.

  1.25

1.93

1.23

  4.08

C.V.

  0.25

0.42

0.65

  0.68

Minimum

  1.06

0.47

0.00

  0.84

Maximum

12.59

13.50

9.21

19.98

about 120 mg DNA and blood frozen at −20°C yields a mean of about 130 mg DNA. Large DNA pellets can be difficult to dissolve in less than 1.0  mL buffer. This difficulty can be noted as instability (CV > 10%) of repeated concentration measurements. Dissolving the DNA pellets in 2.0 mL buffer with rocking at room temperature overnight may improve solubility (CVs around 2%). Yields are generally lower if samples have been poorly mixed in the EDTA tubes at the time of collection so that small clots have formed in the tubes. Such clotting problems are rare if plastic tubes with lyophilised K2EDTA powder are used rather than tubes containing K3EDTA liquid solution (3). Very low can occur in some samples due to pellet loss by the Autopure LS instrument. 3. Commercial filtre paper products intended for nucleic acid collection and designed to bind PCR inhibitors while enabling the release of DNA without the need for proteolytic enzymes or incubation with chemicals are available (4). Using 100 mL of fresh control whole blood with known WBC on Schleicher and Schüll 2992 a 6 mm disc typically yields 13% of the theoretical DNA content with high molecular weight (5). The presence of PCR inhibitors can be minimised by diluting the samples by a factor of ³20. Storage of samples at −20°C reduces DNA degradation and fragmentation and increases success rate in genetic analyses compared to samples stored at room temperature. 4. Addition of a paraffin bead prior to the 80°C incubation facilitates the removal of a solid paraffin disc after cooling. DNA obtained from FFPE tissue is generally