What are PCR Blockers for PCR Clamping Assays

PCR clamping selectively inhibits amplification of a specific DNA sequence, typically a wild-type or normal allele, enabling detection of rare variants or mutations. In a standard PCR reaction, a dominant wild-type sequence can "out-compete" a low-abundance mutation, making it nearly impossible to detect early-stage cancer markers or minor viral variants. PCR clamping enables the detection of low-abundant mutated sequences in pre-cancerous tissue or cells.

PCR blocker oligonucleotide strands that compete with a primer for a specific binding site suppress the amplification of high-abundance "background" DNA, such as mitochondrial DNA in forensic samples.

Selecting the "best" PCR blocker depends entirely on the specific application selected. Typical applications include suppressing wild-type sequences to detect rare mutations, preventing primer dimers, or blocking the amplification of host DNA in metagenomics. These are known as PNA-mediated PCR clamping", "LNA clamp mutation detection", or "Enrichment PCR rare variant."

PCR clamping relies on synthetic nucleic acid analogs that allow the design of "clamps." The most commonly used analogs are Peptide Nucleic Acids (PNAs) or Bridged or Locked Nucleic Acids (BNAs/LNAs), designed to be perfectly complementary to the wild-type sequence.

A well designed clamp overlaps either a primer-binding site or the sequence immediately downstream. Because PNAs and LNAs have a higher thermal stability and binding affinity than standard DNA, the clamp binds to the wild-type DNA more strongly than the PCR primer can. Once the clamp is bound, it physically blocks the DNA polymerase from extending the primer. If a mutation is present in the target sequence, the resulting mismatch significantly lowers the clamp's melting temperature (Tm). The clamp dissociates from the mutated strand, allowing the primer to bind and the polymerase to amplify the rare mutant sequence.

Both PNA and BNA/LNA allow the design of clamping assays: PNA (Peptide Nucleic Acid) has a neutral peptide-like backbone instead of a charged sugar-phosphate backbone. Because there is no electrostatic repulsion between the PNA and the DNA strand, the binding is very stable. DNA polymerases cannot recognize or degrade PNA, nor can they use it as a primer. LNA (Locked Nucleic Acid) is a modified RNA nucleotide where the ribose ring is "bridged" or "locked" in a specific conformation. They are often incorporated into standard DNA primers to increase specificity and Tm, making the reaction extremely sensitive to single-base mismatches.

PCR clamping enables selective diagnostics in oncology for detecting rare somatic mutations, such as KRAS or EGFR, in liquid biopsies or tissue samples where the majority of DNA is healthy, for pathogen detection to identify drug-resistant strains of bacteria or viruses that exist as minor populations alongside wild-type strains, and for genotyping to distinguish between highly similar alleles or detecting single-nucleotide polymorphisms (SNPs) with high precision.

PNA versus BNA/LNA

PNAs differ from DNA molecules in several aspects:

(i) PNA/DNA-hybrids have a higher thermal stability compared with the corresponding DNA/DNA hybrids (∼1°C/base for mixed sequences);

(ii) PNA/DNA hybrids are more destabilized by single base pair mismatches than the corresponding DNA/DNA hybrids; and

(iii) PNAs cannot serve as primer molecules in PCR.

Advantages and Limitations

Orum et al. reported in 1993 a method for the direct analysis of single-base mutations by the polymerase chain reaction (PCR). The method utilizes PNAs (peptide nucleic acids) that recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribo-oligonucleotides (DNA). Because PNAs have a neutral peptide backbone, they bind to DNA with higher affinity than DNA itself, preventing binding by Taq polymerase. The researchers showed that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA targets one of the PCR primer sites. As a result, PCR blockage allows selective amplification and suppression of target sequences that differ by only one base pair. Blocking PNAs can be designed so that the targeted sequence is between the PCR primers.

Thiede et al. in 1996 used PNA-mediated PCR clamping to detect Ki-ras point mutations. This study was one of the first major applications in oncology, showing how to detect rare KRAS mutations in a high background of wild-type DNA, allowing the detection of 1 mutant in 200 normal cells.

PCR clamping is widely used in "Liquid Biopsy" research to find cancer markers in blood.

PNA-LNA mixed methods for clinical diagnostics

Nagai et al. in 2005 reported the use of a Peptide Nucleic Acid-Locked Nucleic Acid (PNA-LNA) PCR Clamp for the sensitive detection of EGFR mutations in non-small cell lung cancer cell lines. In this approach, the PNA clamp primers suppress amplification of wild-type sequences, thereby preferentially enhancing amplification of mutant sequences. LNA probes are specifically employed to detect mutant sequences in the presence of wild-type sequences. 

Tanaka et al. reported a clamping method based on peptide nucleic acids and locked nucleic acids in 2007. This clamping method allows detection of epidermal growth factor receptor (EGFR) mutations in cytological or paraffin-embedded specimens contaminated with normal cells. The method can detect G719S, G719C, L858R, L861Q, and seven different exon 19 deletions in the presence of 100-1000‐fold excess wild‐type alleles. This approach is a refined version that combines PNA and LNA primers to enhance the sensitivity of cancer-causing mutation detection.

LNA-Specific Clamping

Efrati et al. in 2010 developed an LNA-based PCR clamping method that preferentially amplifies mutated over wild-type KRAS. The study evaluated the method's sensitivity using serial dilutions of plasmids carrying wild-type and mutant KRAS fragments and tested it on 60 archived tissue samples of colon carcinoma, comparing results to direct sequencing and high-resolution melting (HRM) methods.

This PCR clamping method allows the detection of as little as 1% mutated DNA in the analyzed sample. Of the 29 KRAS mutations, only 23 (79%) were detected by standard direct sequencing. This LNA-based PCR clamping method is simple and highly sensitive for detecting KRAS mutations.

Ikenaga et al. in 2015 expanded the applicability of LNA-based PCR assays to enable diagnostics of organelles and bacteria in plants. This study investigated the design of oligonucleotides specific for plastids. PCR without LNA oligonucleotides predominantly amplified organelle genes. However, bacterial genes could be predominantly detected using LNA oligonucleotides. The newly designed LNA oligonucleotides specific for plastids have since widened the scope in investigating the community structures of plant-associated bacteria.

Anderson et al. reported, in 2016, an automated version of the RNA ISH technology, RNAscope, adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining, and results are comparable to those of the manual assay. This method is similar to branched DNA-based assays without the use of branched oligonucleotides. The automated duplex RNAscope assay simultaneously detects two biomarkers.

References

Anderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, Kernag C, Kim J, Luo Y, Lamparski H, Park E, Su N, Ma XJ. Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues. J Cell Biochem. 2016 Oct;117(10):2201-8. [PMC]

Dideoxynucleotide chain termination oligonucleotides and their application

Efrati E, Elkin H, Peerless Y, Sabo E, Ben-Izhak O, Hershkovitz D. LNA-based PCR clamping enrichment assay for the identification of KRAS mutations. Cancer Biomark. 2010-2011;8(2):89-94. [PMC]

Ikenaga M, Sakai M. Application of Locked Nucleic Acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures. Microbes Environ. 2014 Sep 17;29(3):286-95. [PMC]

Nagai Y, Miyazawa H, Huqun, Tanaka T, Udagawa K, Kato M, Fukuyama S, Yokote A, Kobayashi K, Kanazawa M, Hagiwara K. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 2005 Aug 15;65(16):7276-82. [PubMed]

Oligo chain terminator modifications

Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 1993 Nov 25;21(23):5332-6. [PMC]

Tanaka T, Nagai Y, Miyazawa H, Koyama N, Matsuoka S, Sutani A, Huqun, Udagawa K, Murayama Y, Nagata M, Shimizu Y, Ikebuchi K, Kanazawa M, Kobayashi K, Hagiwara K. Reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers. Cancer Sci. 2007 Feb;98(2):246-52. [PMC]

Thiede C, Bayerdörffer E, Blasczyk R, Wittig B, Neubauer A. Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping. Nucleic Acids Res. 1996 Mar 1;24(5):983-4. [NAR]