Increase SNP target specificity and use shorter sequences with these high Tm locked nucleic acid probes
Use Affinity Plus™ qPCR Probes for SNP genotyping, transcript variant identification, and improved target detection in challenging samples (FFPE tissue, biofluids). The Affinity Plus bases used in these qPCR probes include up to 6 locked nucleic acid monomers (see the technology page, Locked nucleic acids, for more information). When incorporated into a probe, locked nucleic acids impart heightened structural stability, leading to increased hybridization melt temperature (Tm).
Yakima Yellow is a registered trademark of EliTech Group, and Cy is a registered trademark of Cytiva.
Prices listed include probe sequence (10–25 standard bases with up to 6 Affinity Plus LNA bases), reporter, quencher, and HPLC purification. See the Product Details tab for design tips.
Cy is a registered trademark of Cytiva; Black Hole Quencher (BHQ) is a registered trademark of Biosearch Technologies, Inc.; TEX™ fluorophore is a trademark of ThermoFisher; TYE fluorophores are licensed from ThermoFisher; Yackima Yellow (YAK) is a registered trademark of EliTech Group
Locked nucleic acid bases, such as Affinity Plus monomers, can be incorporated into effective qPCR probes [1–4]. Because locked nucleic acid bases significantly increase Tm, Affinity Plus qPCR Probes can be designed with shorter lengths
than standard qPCR probes. Shorter probes have better quenching and a higher signal-to-noise ratio and are, therefore, more sensitive. More importantly, locked nucleic acid probes offer an improved ability to distinguish mutations or single nucleotide
polymorphisms (SNPs) . An Affinity Plus qPCR Probe can be designed with several locked nucleic acid monomers resulting in a ΔTm of >15°C, which greatly increases
Sequences containing Affinity Plus locked nucleic acid nucleotides provide identical annealing properties as other manufacturers’ locked nucleic acid sequences (Figure 1). The increased hybridization affinity and thus hybridization melting temperature (Tm) results in enhanced sequence stability both in vitro and in vivo.
In comparison to unmodified probe sequences, the higher melting temperature of the locked nucleic acid-containing Affinity Plus qPCR Probes provides better stability in qPCR assays 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.
The Figure 3 cluster plots from 4 genotyping assays demonstrate that locked nucleic acid containing Affinity Plus qPCR Probes generate identical genotyping calls as PrimeTime LNA Probes. The probe sequences for both Affinity Plus and PrimeTime LNA probes used in the assays are shown below in Table 1 (see Figure 3 sequences).
|Name||Sequence (+N = Affinity Plus or locked nucleic acid nucleotide)|
|Figure 1 sequence|
|Affinity Plus sequence||GGTCCT+T+A+CTTGGTG|
|Other locked nucleic acid sequence||GGTCCT+T+A+CTTGGTG|
|Figure 2 sequences|
|M2-2 forward primer||GAGTCACTATGTATAATCAAAAAAACAC|
|M2-2 reverse primer||TGATCCGGTGGGTATATATGG|
|PrimeTime LNA qPCR probes||/56-FAM/TA+T+CAA+T+CA+AAA+CAA+CC/3IABkFQ/|
|M2-2 gBlocks Gene Fragment|
TTGCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGTCACTCCACTCCC ATGTCCCTTGGGAAGGTCTGAGACTAGGGTTAGAGTCACTATGTATAATCAAAAAAACACAC TATATATCAATCAAAACAACCAAAATAGCCATATATACCCACCGGATCAA
|Figure 3 sequences|