Improve assays with customizable oligos and probes containing cost-effective locked nucleic acids

Locked nucleic acids improve SNP genotyping and other hybridization assays

Affinity Plus DNA & RNA Oligonucleotides and Affinity Plus qPCR Probes contain locked nucleic acid bases and are ideal for scientists with applications that require increased hybridization specificity or improved stability in their oligo designs. The locked nucleic acid bases included in these sequences:

• Increase hybrid melting temperature (T_m), and thus, hybridization specificity
• Improve sequence stability and nuclease resistance, in vitro and in vivo
• Enhance SNP and mismatch discrimination
• Allow for design of shorter, more effectively quenched probes

Locked nucleic acid bases impart heightened structural stability to Affinity Plus DNA & RNA Oligos and Affinity Plus qPCR Probes. This enhanced stability is derived from a methylene bridge bond that links the 2′ oxygen to the 4′ carbon of the RNA pentose ring of this modified RNA base (Figure 1).

Figure 1. An Affinity Plus monomer. These modified bases contain a methylene bridge bond between the 2′ oxygen and the 4′ carbon of the pentose ring.

Use Affinity Plus DNA & RNA Oligonucleotides for increased hybridization T_m, stability and nuclease resistance over standard oligonucleotides (Figure 2). In comparison to unmodified probe sequences, the higher melting temperature of the LNA-containing Affinity Plus qPCR Probes provides better stability in qPCR assays, especially with target regions of low GC content. The increased stability also makes possible the use of shorter probe designs, ideal when target regions are limited in size. Figure 2 demonstrates that Affinity Plus qPCR Probes provide the same amplification profile as PrimeTime qPCR LNA probes, with both showing more rapid target amplification vs. unmodified probes.(Review probe sequences used in these assays in Table 1 of the Performance tab on the Affinity Plus qPCR Probes product page.)

Figure 2. Affinity Plus qPCR Probes generate identical amplification data as PrimeTime LNA qPCR Probes. A custom PrimeTime qPCR Assay was designed to detect the M2-2 gene from the Human respiratory syncytial virus A strain. Using a gBlocks Gene Fragment as a target template, the performance of 2 LNA qPCR probes [17-base probes containing 7 LNA monomers at identical positions, manufactured as either Affinity Plus qPCR Probes or PrimeTime LNA qPCR Probes (T_m = 68.1°C)] were compared to the same DNA sequence without lock nucleic acid modifications (T_m = 49.9°C). (A) Amplification curves using the 3 probes reveal that the Affinity Plus and PrimeTime LNA qPCR Probes yield identical results. (B) Threshold cycle (C_q) values comparing reactions with (gBlocks Gene Fragment) or without (NTC) template. Both probes containing LNA nucleotides were able to specifically amplify the template. NTC = no template control.

Adjustable T_m

The change in sequence conformation of Affinity Plus Oligos and qPCR Probes increases probe:target hybridization binding strength, resulting in an enhanced oligo T_m. Unlike other modifications used to enhance oligo T_m (e.g., MGB modifications), the T_m of Affinity Plus DNA & RNA Oligos and Affinity Plus qPCR Probes can be adjusted based on the location and number of modifications added to the oligo. 1–6 locked nucleic acid monomers can be inserted into a single Affinity Plus qPCR Probe sequence, making it possible to design a probe with a ΔT_m >15°C and to optimize sequence T_m for your specific experiment.

Use Affinity Plus DNA & RNA Oligonucleotides for increased hybridization T_m, stability and nuclease resistance over standard oligonucleotides. Up to 20 locked nucleic acid monomers can be inserted into a single Affinity Plus Oligonucleotide to increase binding strength.

Increased specificity and stability

Use Affinity Plus qPCR Probes for SNP genotyping, transcript variant identification, and sensitive target detection in challenging samples, such as FFPE tissue or biofluids. The tighter binding of Affinity Plus qPCR Probes increases their target specificity. Thus, it becomes easier to design probes for challenging sequence regions, such as those with high AT content, or where target areas are short. The relatively stronger binding strength also makes it possible to use shorter probes in your hybridization assays. Shorter probes can be an asset, especially in rare target detection, such as SNP analysis, as shorter probes are more effectively quenched and provide greater mismatch discrimination, thus returning stronger, more accurate signals.

The cluster plots from 4 genotyping assays shown in Figure 3 demonstrate that LNA containing Affinity Plus qPCR Probes generate clear, tightly clustered genotyping calls identical to those produced with PrimeTime LNA Probes. (Review probe sequences used in these assays in Table 1 of the Performance tab on the Affinity Plus qPCR Probes product page.)

Figure 3. LNA probes synthesized as Affinity Plus qPCR Probes generate identical genotyping calls as PrimeTime LNA Probes. Genotyping assays (n = 4) were run using 46 unique Coriell gDNA samples and the same probe sequences synthesized as either Affinity Plus qPCR Probes or PrimeTime LNA qPCR Probes. There were no differences in calls observed between Affinity Plus and PrimeTime qPCR LNA probes. All genotype calls matched previous results (data not shown). All but rs1333049 are ADME (absorption, distribution, metabolism and excretion) assays.

In addition, Affinity Plus sequences provide increased resistance to exonucleases. Thus, Affinity Plus Oligos and qPCR Probes won’t be degraded as easily in vitro or in vivo.

Broader size and probe quencher options, affordable price

IDT offers Affinity Plus qPCR Probes and Oligonucleotides that are more cost-effective while providing identical performance as PrimeTime qPCR LNA Probes, and locked nucleic acid probes and oligos from other vendors. These Affinity Plus locked nucleic acid bases are widely available across different IDT oligo products.

Learn more or order Affinity Plus DNA & RNA Oligonucleotides.

Learn more or order Affinity Plus qPCR Probes.