Open Access Methodology article

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Joseph R Dobosy, Scott D Rose, Kristin R Beltz, Susan M Rupp, Kristy M Powers, Mark A Behlke* and Joseph A Walder

Author Affiliations

Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 5224, USA

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BMC Biotechnology 2011, 11:80  doi:10.1186/1472-6750-11-80

Published: 10 August 2011

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Additional File 1:

Table S1. Synthetic oligonucleotide sequences employed in this study.

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Additional File 2:

Supplemental Methods: Cloning and characterization of Pyrococcus abyssi RNase H2.

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Additional File 3:

Figure S1. Identification of RNase H2 cleavage products by mass spectrometry. The synthetic oligonucleotide substrates shown were examined before and after cleavage by recombinant Pyrococcus abyssi RNase H2 using electrospray ionization mass spectrometry (ESI-MS). Mass spectra and measured masses are shown to the left. Substrates and reaction products with calculated molecular weights are shown to the right. DNA bases are indicated in black upper case and RNA bases are indicated in red lower case letters.

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Additional File 4:

Figure S2. Mg2+ dependence of P.a. RNase H2 activity. 32P-labeled substrate S-rC 14-1-15 was incubated in the absence or presence of 0.25 mU of recombinant P.a. RNase H2 for 20 minutes at 70°C in Mg Cleavage Buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 μg/mL BSA, 0.01% Triton X-100) with varying concentrations of MgCl2 as indicated. Reactions were stopped with the addition of EDTA and cleavage products were separated by denaturing PAGE and visualized by phosphorimaging. The phosphor gel image was quantified and the percent cleavage of substrate (Y-axis) is shown plotted against Mg2+ concentration (X-axis).

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Additional File 5:

Figure S3. Optimization of primer design for rhPCR. Design of blocked-cleavable primers for use in rhPCR was optimized using a 103 base synthetic oligonucleotide target. A single unmodified Forward (For) primer was used with different blocked-cleavable Reverse (Rev) primers and compared for their relative ability to prime a PCR assay. Blocked-cleavable Rev primers used the same sequence as the unmodified control Rev primer, with the addition of a rU base, and were serially extended by adding 2, 3, 4, 5, or 6 DNA bases 3'-to the ribonucleotide. All blocked primers ended in a ddC residue. Following 45 cycles of PCR, products were separated by denaturing PAGE, fluorescently stained and visualized by UV excitation. M = oligo size markers (bases).

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Additional File 6:

Figure S4. Mismatch discrimination using rhPCR with the mismatch positioned at the "-1" position relative to the RNA base. Sixteen synthetic oligonucleotide targets were employed where the base complementary to the single RNA residue in the blocked-cleavable primers was fixed (A, C, G, or T) and the base paired opposite position "-1" immediately 5'-to the RNA base in the primer was varied (A, C, G, or T). Likewise a set of 16 "rDDDDx" blocked-cleavable primers was employed where the RNA base was fixed (rA, rC, rG, or rU) and the base at the "-1" position was varied (A, C, G, or T). The target sequence and primers were otherwise the same as in Figure S3, except that the control non-discriminatory primer was one base shorter on the 3'-end. Assay conditions and calculations of ΔCq values were the same as in Figure 7 in the manuscript. All reactions were run in triplicate.

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Additional file 7:

Figure S5. Mismatch discrimination using rhPCR with the mismatch positioned at the "+1" position relative to the RNA base. Sixteen synthetic oligonucleotide targets were employed where the base complementary to the single RNA residue in the blocked-cleavable primers was fixed (A, C, G, or T) and the base paired opposite position "+1" immediately 5'-to the RNA base in the primer was varied (A, C, G, or T). Likewise a set of 16 "rDDDDx" blocked-cleavable primers was employed where the RNA base was fixed (rA, rC, rG, or rU) and the base at the "+1" position was varied (A, C, G, or T). The target sequence and primers were otherwise the same as in Figure S3. Assay conditions and calculations of ΔCq values were the same as in Figure 7 in the manuscript. All reactions were run in triplicate.

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Additional File 8:

Table S2. Efficiency of rhPCR at different anneal/extend temperatures. Amplification reactions were run in standard format (10 L reactions with 2.6 mU P.a. RNase H2) using 2-step PCR with anneal/extend temperatures of 50oC, 55oC, and 60oC. The SMAD7 SNP assay and “rDDDDx” blocked-cleavable primers were employed, as in Table 2.

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Additional File 9:

Unified additional materials. All additional files are merged to improve convenience when saving or printing these data.

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