Protocol 101

Genomic DNA samples 1a. Cell line DNA - as a source of external reference material, several genomic DNA samples (e.g. KT17) established for the 10th International Histocompatibility Workshops were purchased from Fred Hutchinson Cancer Research Center (Shao et al., 2004). The pre-determined concentration of 200 ng/^L corresponds to ~28,571 genome copies/^L

1b. Patient DNA samples - these samples were extracted from whole blood using the QIAamp DNA Blood Mini kit (Qiagen) (Tang et al., 2002); DNA concentration was initially estimated by optical density at 260 nm; more reliable quantification was done using the PicoGreen dsDNA Assay (Molecular Probes) that stains double-stranded DNA

Digestion of gDNA with EcoR I (New England Biolabs)

2a. EcoR I reaction mix (60 |L each) 10x NE buffer H 6.0 |jL

2b. Incubate sample at 37°C for 4 hr before inactivating enzyme at 65°C for 20 min (best done in a thermocycler with heated lid)

2c. Separate EcoR I-digested gDNA from salt and enzyme by size exclusion, using 96-well filter plate (Millipore) attached to a manifold vacuum

2d. Recover EcoR I-digested gDNA in 120 |l sterile water. The digested DNA is enough for many downstream applications; with an estimated 95% recovery rate, DNA concentration becomes ~45 ng/|L

Digestion of gDNA 3a. BstU I reaction mix (40 |L each)

EcoR I-treated gDNA 20.0 |L (~45 ng/|L) (one-sixth of the total)

Sterile water 15.5 |L

3b. Incubate sample at 60°C for 2 hr

3c. Separate BstU I-digested gDNA from salt and enzyme by size exclusion, using 96-well filter plate (Millipore) attached to a manifold vacuum

3d. Recover BstU I-digested gDNA in 120 |l sterile water for qPCR (DNA concentration becomes ~7 ng/|L)

qPCR in 96-well plates

4a. Prepare qPCR reaction mixture, with each having a total volume of 25 |L:

2x QuantiTect master mix (Qiagen)*

Forward primer (20 |M) Reverse primer (20 |M) 100x fluorescein BstU I-digested gDNA PCR water

*Reagents from several other suppliers do not work consistently without further optimization

4b. Thermocycling in the iCycler® (with heated lid) (BioRad)

Step 1. 95°C, 15 min (to active the HotStarTaq)

Step 2. 35 cycles of 95°C for 20 sec (denaturing), 60°C (annealing, temperature decreasing at 0.2°C per cycle until it reached 58°C) for 30 sec, 72°C for 36 sec (extension), with optics activated after each extension, Step 3. Plot melting curve from 65°C to 95°C

4c. Characterization of qPCR amplicon (optional) by conventional techniques including

• Run PCR products on 2.0% agarose gel to visualize DNA bands (with ethidium bromide staining and UV transillumination)

• Determine the size of qPCR amplicons by comparing with bands in the 100-bp DNA ladder (New England Biolabs)

• Confirm the DNA sequence identity by nested PCR, Southern blot, or direct cycle sequencing

Design and documentation of qPCR assays

5a. For each experiment, the first 10 wells are reserved for duplicates of five serially diluted (e.g. 1:8, 1:64, 1:512, and 1:4096), positive control DNA (high molecular weight genomic DNA or plasmid DNA containing the target sequence) with a known copy number. Other wells are used for testing samples (also in duplicates)

5b. Verify that each qPCR test yields specific amplicons (e.g. 4c) and that all melting curves overlap to form a single peak; qPCR assays containing an internal probe (e.g. TaqMan®) should be used if these conditions are not met

5c. Use the threshold cycle numbers (Ct) of control samples to generate a standard curve in each test, with known input DNA copy numbers transformed to log10 (e.g. Table 2); experiments are deemed reliable if the r2 for the standard curve is greater than 0.95

5d. With DNA samples treated with EcoR I and BstU I, qPCR for MDR1 promoter fragment 2 (no EcoR I site; no BstU I site) was used to quantify the total input DNA copy numbers in each sample (see Figure 10.1)

5e. qPCR for MDR1 promoter fragment 1 (no EcoR I site; 1 BstU I site) and MDR1 intron 1 (no EcoR I site; 6 BstU I sites) defined the DNA copies resistant to BstU I (due to CpG methy-lation)

5f. The ratio of BstU I-resistant MDR1 DNA to total input DNA serves as a crude measure (see discussion in the text and Figure 10.1) of methylation level in test samples

6. Verification of CpG 6a. Design PCR and sequencing primers using methylation by the Pyrosequencing™ Assay Design pyrosequencing Software

6b. Treat gDNA samples with bisulfite to convert unmethylated cytosine to uracil (U), following procedures outlined for the EZ DNA methyla-tion kit (Zymo Research)

6c. Perform pyrosequencing using the Pyro Gold reagents, as recommended by the manufacturer (Biotage); proportion of methylcytosine (mC) is calculated automatically in the allele frequency mode

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