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 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 PCR blocker is mRNA 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 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 result.
We offer mPNA and pPNA as catalog item for this purpoase. 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 size ($335 each), which is good for 250~2,000 reactions, and 50 nmole size ($475 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.
If you want to use 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.
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 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.
Globin reduction PNA is composed of following 4 PNAs that are specific to alpha and beta globin mRNA. It works for both human and mouse globin mRNA.
PNA1: k-TAA CGG TAT TTG GAG-k
PNA2: k-GTA GTT GGA CTT AGG-k
PNA3: k-GCC CTT CAT AAT ATC-k
PNA4: k-ATC CAG ATG CTC AAG-k
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A set of 4 PNA oligos against human globin,
A set of 4 PNA oligos against human globin