What are the best PCR Blockers

The most effective chemical and structural blockers currently used in molecular biology are:

Peptide Nucleic Acids (PNAs)

PNAs are the "gold standard" for sequence-specific blocking, featuring a neutral peptide-like backbone instead of the negatively charged phosphodiester backbone. Because they lack electrostatic repulsion, PNAs bind to complementary DNA with much higher affinity and specificity than DNA-DNA duplexes. In PNA-clamping, a PNA probe that matches a wild-type sequence will remain bound even at high temperatures, physically preventing the DNA polymerase from extending past it, allowing the selective amplification of mutant sequences.

Locked Nucleic Acids (LNAs)

LNAs contain a methylene bridge that "locks" the ribose ring in the C3'-endo conformation. The resulting structural rigidity significantly increases the duplex's melting temperature (Tm). An LNA-modified blocker can be much shorter than a standard DNA probe while maintaining a high specificity. LNA-based allele-specific PCR allows discriminating between sequences that differ by only a single nucleotide.

C3-Spacer (Propyl Group) or Phosphate Blocks

The 3’-end of a blocker can be modified to prevent a specific primer or probe from being extended by the polymerase. Adding a 3'-C3 spacer, 3'-Phosphate, or even a dideoxycytidine (ddC) prevents the polymerase from adding the next nucleotide. This type of modification is best for dual-probe assays or Sanger sequencing, where the probe hybridizes to a target for detection, such as in a TaqMan assay, without acting as a primer.

Morpholinos (PMOs)

While more commonly used in gene knockdown, Phosphorodiamidate Morpholino Oligomers (PMOs) also enable the design of blockers for specialized diagnostic assays. Similar to PNAs, they have a nonionic backbone, are highly resistant to nucleases, and bind strongly to target sequences.

Applications requiring blockers with high stability in complex biological samples, where exonucleases might degrade standard DNA blockers, can use PMOs.

Non-Extendable "Double-Strand" Blockers

Sometimes, the best blocker is simply an excess of a complementary oligonucleotide that is modified to be non-extendable. A "blocker" strand that competes with primers for a specific binding site suppresses amplification of high-abundance "background" DNA, such as mitochondrial DNA in forensic samples.

Selection of PCR Blockers

Note: For the design of a blocker for PNA-clamping, ensure the blocker Tm is significantly higher, usually >10°C, than the primer to ensure that the blocker is firmly bound to the template before the primers attempt to bind and extend.