gene_expression

PrimeTime® BNA (bicyclic nucleic acid) qPCR Probes

Increased sensitivity for distinguishing DNA base-pair mismatches

A bicyclic nucleic acid is a modified RNA nucleotide that when incorporated into an oligonucleotide sequence, imparts structural rigidity and improves base stacking for greater Tm. This modification is not compatible with PCR, because modified bases are not extendable by Taq polymerase.

  • Increase assay specificity over traditional 5′ nuclease probe sequences
  • Create more nuclease-resistant oligonucleotide probes
  • Choose from a wide variety of reporter and quencher combinations
  • Get started sooner: standard BNA probes ship in 4–6 business days

Ordering

Prices listed include probe sequence (up to 25 bases in length), up to 6 BNA base insertions, reporter, quencher, and HPLC purification. Probes are shipped in 4–6 business days.

Submit custom designs directly (see Product details for design considerations), or for design assistance, email applicationsupport@idtdna.com (design fees may apply).

PrimeTime BNA qPCR Probes

Dual-labeled, 5′ nuclease DNA probes incorporating 1–6 BNA bases for increased hybridization Tm and binding specificity.

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' 6-FAM™3' Black Hole Quencher® 1250 nmole8 nmoles$780.00 SGD
1 µmole20 nmoles$1,110.00 SGD
3' Iowa Black® FQ250 nmole8 nmoles$645.00 SGD
1 µmole20 nmoles$900.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' Cy3™3' Black Hole Quencher® 2250 nmole8 nmoles$860.00 SGD
1 µmole20 nmoles$1,220.00 SGD
3' Iowa Black® RQ-Sp250 nmole8 nmoles$715.00 SGD
1 µmole20 nmoles$1,000.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' Cy5™3' Black Hole Quencher® 2250 nmole8 nmoles$875.00 SGD
1 µmole20 nmoles$1,240.00 SGD
3' Iowa Black® RQ-Sp250 nmole8 nmoles$730.00 SGD
1 µmole20 nmoles$1,020.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' TEX 6153' Black Hole Quencher® 21 µmole20 nmoles$1,575.00 SGD
250 nmole8 nmoles$1,130.00 SGD
3' Iowa Black® RQ-Sp1 µmole20 nmoles$1,435.00 SGD
250 nmole8 nmoles$1,025.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' TYE™ 5633' Black Hole Quencher® 21 µmole20 nmoles$1,530.00 SGD
250 nmole8 nmoles$1,095.00 SGD
3' Iowa Black® RQ-Sp1 µmole20 nmoles$1,385.00 SGD
250 nmole8 nmoles$990.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' TYE™ 6653' Black Hole Quencher® 21 µmole20 nmoles$1,530.00 SGD
250 nmole8 nmoles$1,095.00 SGD
3' Iowa Black® RQ-Sp250 nmole8 nmoles$990.00 SGD
1 µmole20 nmoles$1,385.00 SGD

5' Reporter DyeQuencherSynthesis ScaleMin GuaranteePrice
5' HEX™3' Black Hole Quencher® 1250 nmole8 nmoles$810.00 SGD
1 µmole20 nmoles$1,140.00 SGD
3' Iowa Black® FQ250 nmole8 nmoles$675.00 SGD
1 µmole20 nmoles$940.00 SGD

IDT has licensed from Isis Pharmaceuticals the rights to manufacture BNA oligonucleotides for life sciences research and development activities, excluding therapeutic, diagnostic, in vivo, and mass spectroscopy applications. BNA oligonucleotides are not licensed for resale or transfer to third parties. Diagnostic licenses for BNA oligonucleotides are available from Isis Pharmaceuticals.

BNA bases have a modification to the ribose backbone that locks the base in the C3′-endo conformation, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also relatively nuclease resistant.

BNAs can be incorporated into dual-labeled probes for use in 5′ nuclease assays. Because BNA bases significantly increase Tm, PrimeTime BNA Probes can be designed with shorter lengths than standard dual-labeled probes. Shorter probes are more effectively quenched and have a higher signal-to-noise ratio; therefore, they are more sensitive.

More importantly, these probes offer an improved ability to distinguish mutations or single nucleotide polymorphisms (SNPs). A DNA dual-labeled probe typically has a ΔTm of ~5°C between a perfect-match and mismatched hybridization. In contrast, a BNA dual-labeled probe can have a ΔTm of >15°C, greatly increasing accuracy of allele determination in real-time PCR or other methods that use differential hybridization to distinguish polymorphisms.

Design considerations

Tm calculations can be made using the OligoAnalyzer® Tool. The LNA notation (+T, +A, +G, +C) to estimates Tm or mismatch discrimination potential of BNA bases. BNA probe design points to consider include:

  • Insertion of a BNA base into a DNA oligo can increase the Tm by 3–6°C, depending on sequence context. However, there are some sequence-specific designs involving G-T mismatches where BNA bases may impair specificity.
  • We recommend including up to 6 BNA bases in a BNA dual-labeled probe:
    • BNA bases should be placed at the SNP site and adjacent bases.
    • The SNP should be positioned in the center of the probe, if possible.
    • Additional BNA bases can be incorporated toward the 3′-end of the probe to adjust Tm, as needed.
  • The relative binding affinity (Tm) of BNA bases are BNA:BNA > BNA:DNA > DNA:DNA. Therefore, it is important to examine the probe sequence for self-dimer and hairpin formation and minimize designs that allow BNA:BNA pairing.

For additional assistance with design of PrimeTime BNA Probes, email applicationsupport@idtdna.com (design fees may apply).

Related reading

  1. Owczarzy R, You Y, et al. (2011) Stability and mismatch discrimination of locked nucleic acid−DNA duplexes. Biochem 50:9352–9367.
  2. You Y, Moreira BG, et al. (2006) Design of LNA probes that improve mismatch discrimination. Nucl Acid Res 34(8):e60. doi:10.1093/nar/gkl175

OligoAnalyzer Tool

Identifies oligonucleotide properties, including melting temperature, hairpins, dimers, and mismatches

UNAFold Tool

Performs secondary structure analysis