PCR Blockers

PNA oligo can be used as a sequence-specific PCR blocker because PNA probes have strong binding affinity and specificity to its target DNA and not being recognized by DNA polymerase as a primer. This method is also called PNA Clamping. PCR Blocker or PNA Clamping is an efficient way to correct amplification bias and sequencing errors.

This is one way to achieve SNP specific genotyping of the gene of interest, such as mutant screening.

One of the examples of this approach is globin reduction PNA. Using a mixture of PNA that is specific for human blood gamma globin RNA, the PNA suppresses the amplification of the globin RNA during cDNA synthesis for microarray application.

The other popular application of PNA as a PCR blocker is mPNA and pPNA. These two PNA oligos are made against chloroplast and mitochondrial 16S sequences from diverse plant species. They are proven to be a useful tool in megagenomics.

PNA oligo can be custom made for each application. For more detail, please refer Custom PNA Oligos.

For taxonomical classification of metagenomics, 16S/18S and 28s rRNA sequencing is commonly used. One of the common problems encountered is the contamination of 16S sequences originating from the host's genome. Plastid or mitochondria can account for >80% of the sequences obtained. Although modification of the bases in the 'universal' amplicon primers can mitigate amplification of the contamination, this can also lead to bias. PNA clamps designed to suppress plant host plastid and mitochondrial 16S contamination can reduce the bias and result in more accurate sequencing data.

We offer mPNA and pPNA as catalog items for this purpose. The sequence of mPNA (to block mitochondria contamination) is ggcaagtgttcttcgga and pPNA (to block chloroplast contamination) sequence is ggctcaaccctggacag.

They are available as 25 nmole ($395 each), which is good for 250~2,000 reactions, and 50 nmole ($550 each), which is enough for 500~4,000 reactions in 50 ul PCR volume. For starters, 250 uM each of mPNA and pPNA is recommended for 50 ul PCR reaction. For more complete PCR blocking, you can use up to 2 uM of mPNA and pPNA. In general, the more PCR blockers are added, the better the clamping effect is.

Please see the reference below for more details about mPNA and pPNA sequences and applications.

  • Practical innovations for high-throughput amplicon sequencing. Lundberg DS et al. (2013) Nat Methods 10:999-1002

  • We also offer ITS PNA (sequence: CGAGGGCACGTCTGCCTGG) to block amplification from the internal transcribed spacer (ITS). It is $425 for 25 nmole (IP01-25) and $585 for 50 nmole (IP01-50).

    If you want to use a different sequence of PCR blocker for other species, we can provide them as custom made PNA oligos. Please check Custom PNA Oligos for more details.

    Usually, mPNA and pPNA probes are in stock and shipped out the same day if the order is placed by 5 pm EST. For Globin reduction PNA, the lead time is about one week.
    Download a protocol for mPNA and pPNA PCR Blocker. 

    Globin mRNA is a majority of total mRNA in blood cells (over 70%) and can potentially reduce the sensitivity of non-globin mRNA.

    Globin Reduction PNA is a novel, non-enzymatic technology that removes the majority of alpha and beta globin mRNA from total RNA preparations derived from whole blood. PNA oligomers can be effectively used as a clamp by specifically blocking globin mRNA during the process of reverse transcription and resulting in specific PCR amplification of the target non-globin mRNA for your analysis.

    The 3 nmole kit includes sufficient reagents for 250 reactions of 5 ug RNA preps.

    Download protocol for Globin Reduction PNA Kit.

    Globin reduction PNA is composed of the following 4 PNAs that are specific to alpha and beta globin mRNA. It works for both human and mouse globin mRNA.

  • The sequences of G2001, GR PNA-L:

    1. Practical innovations for high-throughput amplicon sequencing. Lundberg DS et al. (2013) Nat Methods 10:999 -1002.
    2. Identifying the plant-associated microbiome across aquatic and terrestrial environments: the effects of amplification method on taxa discovery. Jackerel SL et al (2017) Molecular Ecology Resources 1755-0998.12645.
    3. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism. Hong CS et al (2016) Mol Ther Nucleic Acids. 5(4): e314.
    4. DNA Clutch Probes for Circulating Tumor DNA Analysis. Das J et al. (2016) J. Am. Chem. Soc. 138 (34):11009–11016.
    5. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches. Khodakov D et al. (2016) Adv Drug Deliv Rev. 105(Pt A):3-19.
    6. Rapid Intraoperative Molecular Characterization of Glioma. Shankar GM et al. (2015) JAMA Oncol. 1(5):662-667.
    7. Fractal circuit sensors enable rapid quantification of biomarkers for donor lung assessment for transplantation. Sage AT. (2015) Sci Adv. 1(7):e1500417.
    8. Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples. Choi I et al (2014) BMC Genomics. 15:954.
    9. Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes. Taback B et al. (2004) Int J Cancer. 111(3):409-14.
    10. Single base pair mutation analysis by PNA directed PCR clamping. Orum H et al (1993) Nuc Acids Res 21(23):5332-5336.
    11. Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel Leptocephali. Terahara T et al (2011) pLOS One 6(11):e25715.
    12. High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping. Urata M et al (2004) Clin Chem 50 (11)2045-2051.
    13. Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR. Bender M et al (2007) Biotechniques 42(5):609-1.
    Cat No
    GR PNA-S

    A set of 4 PNA oligos against human globin

    3 nmole
    GR PNA-L

    A set of 4 PNA oligos against human globin

    10 nmole

    mitochondria rRNA blocker, ggcaagtgttcttcgga

    25 nmole

    mitochondria rRNA blocker, ggcaagtgttcttcgga

    50 nmole

    chloroplast rRNA blocker,   ggctcaaccctggacag

    25 nmole

    chloroplast rRNA blocker,   ggctcaaccctggacag

    50 nmole

    ITS rRNA blocker,   cgagggcacgtctgcctgg

    25 nmole

    ITS rRNA blocker,   cgagggcacgtctgcctgg

    50 nmole
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