技术概览：
rhPCR.PNG
Benefts of rhPCR
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Elimination of primer dimers
图2、rhPCR有效去除引物二聚体
未添加RNase H2的情况下，在阴性样本中PCR扩增，使用未修饰的引物仅获得不同大小的非特异性片段，使用修饰过的引物不产生任何片段；在阳性样本中扩增，使用未修饰的引物获得少量目标片段和大量不同大小的非特异性片段，使用修饰过的引物不产生任何片段；在阳性样本和相似样本组成的混合样本中扩增，使用未修饰的引物获得少量目标片段和大量不同大小的非特异性片段，使用修饰过的引物不产生任何片段。
添加RNase H2的情况下，在阴性样本中PCR扩增，使用未修饰的引物仅获得不同大小的非特异性片段，使用修饰过的引物不产生任何片段；在阳性样本中扩增，使用未修饰的引物获得少量目标片段和大量不同大小的非特异性片段，使用修饰过的引物仅产生目标片段；在阳性样本和相似样本组成的混合样本中扩增，使用未修饰的引物获得少量目标片段和大量不同大小的非特异性片段，使用修饰过的引物仅产生目标片段。
- Improved Performance of Multiplex Reactions
图3、修饰引物改善多重PCR表现。泳道1:15对引物单独扩增，之后混样（对照）；泳道2：用rhPCR（修饰引物）进行15x的PCR；泳道3：未修饰引物进行15xPCR反应。
可以看到修饰引物多重扩增的片段分布与对照的基本一致，仅在不同片段的总量上有差异；未修饰引物多重扩增的片段与对照的相差很大。说明rhPCR确实改进了多重PCR反应的表现。
- Improved Specifcity: Discriminating Closely-Related Genes
图4、rhPCR提升特异性
rhPCR能够有效区分目标区域和相似区域，可有效避免肿瘤研究中人与小鼠的交叉反应。未修饰的引物分别对人的cDNA和鼠的cDNA进行荧光定量PCR，发现两者中均存在阳性扩增；修饰的引物分别对人的cDNA和鼠的cDNA进行荧光定量PCR，发现仅在人的样本中纯在阳性扩增。
- Detection of single base pair mismatches with rhPCR
Figure 7 Mismatch discrimination using blocked-cleavable primers with an RNA:DNA base-pair mismatch versus standard unmodified
allele-specific PCR primers.
The effects of mismatches at the “0” positions on the rhPCR assay .
单碱基错配在核糖核苷酸处时，rhPCR（一端常规引物，一端修饰引物）相比AS-PCR能够更好的区分。
Allele-specific PCR using the standard unmodified primers showed ΔCq values ranging from 1.2 to 10.9 with an average of 5.4.
The rhPCR assay showed a much higher level of discrimination with ΔCq values ranging from 5.3 to 14.9 and an average cycle delay of 10.9；All twelve mismatches were easily detected with ΔCq values greater than 5 cycles.
The effects of mismatches at the “-1” and “+1” positions on the rhPCR assay were also studied.
Mismatch discrimination varied from 0.8 to 16.1 cycles for the “-1” series, with an average ΔCq value of 7.7.
For the “+1” series, ΔCq varied from 0.2 to 13.8 cycles, with an average value of 6.6.
Although mismatch discrimination varied with sequence context, overall the greatest specificity was achieved by placing the mismatch opposite to the RNA base (position “0”).
- Improved Specifcity: SNP Detection
图5.rhPCR检测SNP位点
rhPCR能够更好的识别错配，提高SNP分型的精度
Standard allele-specifc primers resulted in mismatch discrimination of <2.0 cycles. Using the blocked-cleavable primers, discrimination of the rU:G (black curves) and rC:A (blue curves) mismatches were 12.6 and 12.1 cycles, respectively (图 5).
图6、rhPCR很好的区分rs4939827/SMAD7位点纯合和杂合情况（实时荧光和end point法）
The correct genotype (homozygous T/T, heterozygous T/C or homozygous C/C) was easily called (图6) in real-time (45 cycles) and end-point (35 cycles) PCR formats.
- Rare Allele Dtection
表1、rhPCR检测稀有位点
无论在是否有背景噪音的情况下，rhPCR均能检测到稀有位点。
- RNase H2 can also be used to increase the specificity of
DNA ligation assays (data not shown).
参考资料：
- rhpcr-white-paper
- Dobosy J, Rose S D, Beltz K, et al. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers[J]. BMC Biotechnology, 2011, 11(1): 80-80.
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