Introduction

Heterozygous deletions and duplications typically account for 10% to 15% of mutations. In some situations, the contribution of heterozygous deletions is even greater [e.g., more than one-third of the pathogenic familial BRCA1 mutations in breast/ovarian cancer in northeast Italy (1) and 55% of mutations in MLH1 and MSH2 genes in hereditary nonpolyposis colorectal cancer in the The Netherlands are deletions (2)].

Heterozygous deletions can be detected by multiplex or duplex PCR (3,4,5,6,7,8). However, two major problems exist with these techniques: primer-dimer formation and false priming. As a result, assay optimization becomes much more difficult as the number of primers increases. Multiple-step dosage methods are available. In multiplex amplifiable probe hybridization (MAPH), short multiple amplifiable probes that are recovered quantitatively after hybridization to genomic DNA are amplified simultaneously by PCR with an additional pair of primers included to assess the copy number of up to 40 genome loci (9). In multiplex ligation-dependent probe amplification (MLPA), pairs of oligo-nucleotides hybridize to adjacent sites on the genomic DNA and are subsequently ligated. The ligated probes are then amplified by PCR with an additional pair of primers, because the probes have identical end sequences (10). Both methods require substantial optimization for each assay.

Pyrophosphorolysis-activated polymerization (PAP) is a novel method for nucleotide acid amplification (11,12). In PAP, pyrophospho-rolysis of 3′ blocked oligonucleotides (P*) is followed by extension ((Figure 1)A). Since the P*s can remain inert until activated, upon annealing to their perfectly matched template (12), multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP) reactions are predicted to proceed with minimal primer-dimer formation or false priming.

Figure 1.

Herein, we applied PAP in a multiplexed and quantitative way to detect deletions in the human factor IX gene ((Figure 1)B). Seven target exons of the factor IX gene are amplified quantitatively, along with one endogenous internal control from the ATM gene, in order to determine copy number. In a blinded analysis, we accurately detected all heterozygous deletions in the factor IX gene.

Materials and Methods

Genomic DNA Samples

Genomic DNA was isolated from whole blood and stored in Tris-EDTA (TE) buffer. Carriers of deletions in the factor IX gene were previously determined using PCR-based methods in this laboratory. The concentration of DNA samples was measured by UV spectrophotometry at 260 nm. The DNA samples were diluted to a working concentration of 30 ng/µL in TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.0). Treatment with proteinase K (2 mg/ mL; Roche, Indianapolis, IN, USA) for 2 h at 65°C and then incubation at 90°C for 10 min greatly improved the consistency among the samples (13,14).

Synthesis of 3′ Blocked Oligonucleotides

Each P* was 30 nucleotides long and contained a dideoxynu cleotide at its 3′ terminus ((Table 1)). F9(20640)30D, F9(23450)30D, and F9(33019)30D, each terminating with a dideoxycytosine nucleotide at the 3′ terminus, were chemically synthesized in 3′ to 5′ direction using phosphoramidite chemistry (Integrated DNA Technologies, Coralville, IA, USA). For all the other P*s, a dideoxyguaine nucleotide was added to the 3′ terminus of a 2′-deoxynucleotide oligonucle otide by terminal transferase. The reaction was performed in a total volume of 30 µL and contained the following mixture: 100 mM potassium cacodylate, pH 7.2, 2.0 mM COCl₂, 0.2 mM dithiothreitol (DTT), 2 nmol of the oligonucleotide, 2.4 mM ddNTP (the molar ratio of the 3′-OH terminus to ddNTP was 1:30; Roche), and 100 U of terminal transferase (Invitrogen, Carlsbad, CA, USA). The reaction was incubated at 37°C for 6 h and then stopped by adding EDTA to a 5 mM final concentration. After desalting using a Centri-Spin 20 column (Princeton Separations, Adelphia, NJ, USA), P* was purified by preparative 7 M urea/18% polyactylamide gel electrophoresis (PAGE) with 30 mM triethanolamine/tricine buffer (pH 7.9 at 25°C) at 45°C for 14-16 h (15,16). The blocked 30-mer was separated from the unblocked 29-mer by 3% difference in mobility on the gel (35 cm length × 1.1 mm thickness). The blocked 30-mer was cut from the gel with a razor, placed in a microcen trifuge tube, and crushed; 1 mL water was then added, and the tube was shaken at 250 rpm at 37°C overnight. The supernatant was centrifuged for two rounds each for 2 h at 7500×g with a YM-3 Amicon^® Centricon^® column (Millipore, Bedford, MA, USA). The recovered P* was determined by UV absorption at 260 nm.