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Robust dosage PCR (RD-PCR), a duplex and quantitative PCR, detects large heterozygous deletions and duplications in genomic DNA samples. RD-PCR amplifies an endogenous internal control and a target locus. Two of six RD-PCR assays behaved anomalously due to lower yields specific to the targets. The variability was eliminated by heat treatment of the genomic DNA samples in 2× TE solution at 90°C for 10 min. Heat treatment improves the utility of RD-PCR and may be generally helpful in multiplex PCR quantitation. The mechanism by which heat treatment eliminates inter-individual variation is unclear. The variability is not associated with DNA extraction methods, RNA contamination, or solution protein contamination, but may reflect inhibition from tightly bound chromatin proteins.
Quantitation is essential for detection of large genomic duplications or deletions. Quantitative PCR has been achieved with external controls (1,2,3), competitive PCR with an internal dosage control (4,5,6), and multiplex PCR (7,8,9,10,11).
Quantitation of gene dosage remains a challenge, and it is known that multiplex PCR is sensitive to DNA quality (12,13). Impurities in DNA samples can exert their effects by altering the efficiency of amplification in the exponential phase and the plateau phase of one or more segments. In addition, point mutations downstream of the primer within the amplified region can inhibit amplification (14,15).
Robust dosage PCR (RD-PCR), a duplex quantitative PCR, was developed recently for the rapid and accurate detection of large heterozygous deletions and duplications (16,17). RD-PCR amplifies an endogenous internal control and target locus. The internal control has a known gene copy number per cell, while the target has an unknown dosage number per cell. The ratio of yield (ROY) is directly proportional to the ratio of the two input templates, so the copy number of the target gene can be obtained from the ROY and the known copy number of the internal control.
When multiple RD-PCR assays were developed, significant inter-individual variation occurred in two regions of the MECP2 gene. This variation was easily eliminated by heating the genomic DNA samples in 2× TE solution (20 mM Tris-HCl, 1 mM EDTA, pH 8.0 at 25°C) at 90°C for 10 min.
Materials and Methods Genomic DNA SamplesGenomic DNA was prepared from peripheral blood by the Puregene® DNA Isolation kit (Gentra, Minneapolis, MN, USA), the standard phenol/chloroform protocol (18), or the Super Quick Gene DNA extraction kit (Analytical Genetic Testing Center, Denver, CO, USA). The samples were extracted and stored at -20°C for 0–10 years.
Sample PretreatmentDNA concentrations were measured by UV spectrophotometer at 260 nm and adjusted to a working concentration of 30 ng/µL in 2× TE. Note that 1× TE eliminates the inter-individual variation, but decrease the yields. DNA samples were pretreated at 90°C for 10 min and then placed at 4°C until the RD-PCR assays were performed. Other pretreatments were also performed (see Results and Discussion).
RD-PCR AssaySix assays were designed ((Table 1)) according to Liu et al. (16) except for a shorter 5′ universal tail (5′-GGCCAAGTGT-3′). Each assay contained a target and an internal control segment. The assays were divided into two groups (group I and II) depending on whether the ATM or FUT gene was used as the autosomal control segment. Group I had four assays with GC contents ranging from 36.6% to 55%; group II had two assays with GC content of 61% and 56%.
Table 1. List of Primer Pairs and PCR Segments
aMECP2, the methyl CpG binding protein 2 gene, Xq28; FVIII, the coagulation factor VIII gene, Xq28; ATM, the ataxia telangiectasia mutated gene, 11q22-q23; FUT2, the fucosyltransferase 2 gene, 19q13; Tm, melting temperature.
bThe sequence of the 3′ sequence-specific region is shown. A 10-nucleotide universal tail (5′-GGCCAAGTGT-3′) is attached to the 5′ end of each primer.
cThe core PCR segment does not include the tails.
dExon 12 of the ATM gene is the internal control of assays 1–4; the FUT2 is the internal control of assays 5–6.
The PCR mixtures contained a total volume of 25 µL: 1× Expand™ High Fidelity buffer #3 (Roche Applied Science, Indianapolis, IN, USA), 4.5 mM MgCl2, 200 µM of each dNTP for group I, or 3.0 mM MgCl2 and 150 µM dGTP/50 µM deaza-dGTP, 200 µM of each other dNTPs, and 10% dimethylsulfoxide (DMSO) for group II, 0.1–0.2 µM of each pair of primers, 1 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), and 1 U Platinum Taq DNA polymerase High Fidelity (Invitrogen), 0.5 µg of bovine serum albumin (BSA), and 60 ng of genomic DNA. The cycling entailed denaturation at 94°C for 15 s, annealing at 55°C for group I or 65°C for group II for 30 s, and elongation at 72°C for 1 min for 23 cycles with a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA).
QuantitationTwelve microliters of PCR product were electrophoresed through a standard 2% agarose gel. Gels were stained in 0.2 µg/mL ethidium bromide for 1 h and scanned by the Typhoon™ 9410 Imager (Amersham Biosciences, Piscataway, NJ, USA) with the following parameters: focal plane = +3 mm; laser wavelength = green, 532 nm; emission filter = 610 BP 30; photomultiplier voltage = 600 V; pixel size = 100 µm; and sensitivity = normal.
