Dual-Labeled DNA Probes

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LNA Probes

 

The LNA Fluorescent Probe Advantage

Increased thermal stability and hybridization specificity
Introducing LNA chemistry into a real-time quantitative PCR probe increases thermal duplex stability² and improves specificity of probe hybridization to its target sequence³ . As such, background fluorescence from spurious binding is reduced and the signal-to-noise ratio is increased.

The LNA monomer chemical structure enhances the stability of the hybridization of the probe to its target. As a result, the duplex melting temperature (T_m) may increase by up to 8°C per LNA monomer substitution in medium salt conditions-compared to a DNA fluorescent probe for the same target sequence-depending on the target nucleic acid⁴. This increase in hybridization creates a significant broadening in the scope of assay conditions and allows for more successful single-tube multiplexing⁵.

Further, it is possible to optimize the T_m level and the hybridization specificity through specific placement of the LNA base(s) in the probe design⁶.

Figure 1: An increase in the number of LNA bases within a Q.PCR probe increases T_m value

Probe Sequence (5' ->3')	LNA base	Tm*	Tm	Tm/LNA
dG dT dG dA dT dA dT dG dC	-	29ºC	-	-
dG + T dG dA + T dA +T dG dC	3	44ºC	15ºC	+5ºC
+ G + T + G + A + T + A + T + G + C	9	64ºC	35ºC	+3.9ºC

* T_m of duplex between probe and its complementary DNA sequence
Note: + symbol denotes the LNA base.

More accurate gene quantitation and allelic discrimination
The ability of Q.PCR probes to readily discriminate between SNPs, the most abundant form of genetic variation, is greatly enhanced by the incorporation of LNA bases^7-9.

Figure 2: LNA dual-labeled fluorogenic probes discriminate better than DNA dual-labeled fluorogenic probes in SNP genotyping analysis¹².

Key
Pink plots: Mutant-type DNA analysis with LNA mutant probe (16 mer with 3 LNA bases)
Green plots: Mutant-type DNA analysis with DNA mutant probe (25 mer)
Purple plots: Wild-type DNA analysis with LNA mutant probe (16 mer with 3 LNA bases)
Red plots: Wild-type DNA analysis with DNA mutant probe (25 mer)

Utilizing LNA for allelic discrimination is an extremely reliable and effective means for SNP-calling in genotyping applications. The presence of a single base mismatch has a greater destabilizing effect on the duplex formation between a LNA fluorescent probe and its target nucleic acid, than with a conventional DNA fluorescent probe. Shorter probes incorporating LNA bases can be used at the same temperatures as longer conventional DNA probes.

Easier and more flexible probe designs for problematic target sequences
Due to LNA's enhanced hybridization characteristics and then significant T_m contribution, LNA-containing Q.PCR probes can be synthesized to be shorter, allowing flexibility in design while still satisfying assay design guidelines. As such, certain design limitations that cannot be overcome with standard DNA probe chemistries can now be reduced or eliminated.

In addition, by using LNA fluorescent probes, shorter probes can be designed to address traditionally problematic target sequences, such as AT- or GC-rich regions. Also, the design of probes for querying difficult or inaccessible SNPs, such as the relatively stable G:T mismatch, is greatly facilitated by LNA.

For example, AT-rich Q.PCR probes often need to be over 30 bases long (and sometimes over 40 bases) to satisfy amplicon design guidelines–but may still perform poorly. With LNA fluorescent probes, the selective placement of LNA base substitutions facilitates the optimal design of highly specific, shorter probes that perform well, even at lengths of 13 to 20 bases!

Compatible with real-time PCR platforms and end-point analytical detection instruments
LNA fluorescent probes are compatible with real-time PCR platforms and end-point analytical detection instruments, depending on the excitation/emission wavelengths of the dyes and the equipment. This gives you the freedom to work with the instrumentation and reagents platform of your choice under universal cycling conditions. No additional capital expenditure for specialized equipment is required.

LNA monomers are incorporated by the same standard phosphoramidite chemistry as DNA and RNA monomers
LNA-containing oligonucleotides are amenable to the same synthesis and modification protocols as DNA and RNA bases. They are soluble in standard buffers and water, and follow basic Watson-Crick base-pairing rules¹⁰.

Features/Benefits of LNA Fluorescent Probes Higher Tm by LNA contribution increases thermal stability and hybridization specificity More accurate gene quantitation and allelic discrimination improves SNP detection assays Shorter LNA probes enable easier and more flexible probe designs for problematic target sequences Compatible with real-time PCR platforms and end-point analytical detection instruments - no additional capital investment is required Incorporation of LNA monomers by standard phosphoramidite chemistry allows the use of the same synthesis/modification protocols as DNA and RNA bases10.

 

Specifications of LNA Fluorescent Probes for Real-Time Quantitative PCR

Quality control	All of our oligonucleotides undergo vigorous process monitoring and strict quality control. Length and labeling are systematically controlled by PAGE or MALDI-TOF mass spectrometry analysis. Quantity is determined by UV Absorbance at 260 nm. Fluorescent probes are systematically quality-controlled by RP-HPLC. Additionally, the ratio between dye absorbance (nm) and DNA/RNA absorbance (nm) is calculated for the main product peak. This ratio defines the purity of the labeled probe, demonstrating that no contamination from partially labeled or unlabeled probes exists.
Purification	Fully deprotected and desalted. Purified by PAGE or RP-HPLC
Length	7 to 40 mers (excluding labels and/or modifiers)
Bases	DNA/LNA chimeras: • LNA (+A, +C, +G or +T) (+ denotes the LNA base) • DNA (A, C, G or T) LNA LightCycler probes can contain up to 6 LNA bases LNA dual-labeled fluorogenic probes and LNA molecular beacons can contain up to 8 LNA bases
Backbone	Phosphodiester bond
Format	Delivered in dry form, in opaque tubes
Turn-around time (TAT)	• LNA LightCycler probes: 6 working days • LNA dual-labeled fluorogenic probes: 7 working days • LNA molecular beacons: 7 working days Turn-around time is dependent upon successful QC validation and does not include shipping time. Please check with your Sigma Custom Products representative for local delivery times.
Storage and stability	Although oligonucleotides can remain stable in solution at 4°C for up to two weeks, Sigma Custom Products strongly recommends storage at -20°C. Repetitive freeze-thaw cycles should be avoided by storing as aliquots. Storing at concentrations above 20 µM in TE Buffer is recommended. Oligonucleotides with fluorescent labels should be protected from light. Sigma guarantees its oligonucleotides for six months, when stored under the above conditions.
Shipment	Shipped by express delivery, dry, in individual, opaque tubes
Oligonucleotide Technical Data Sheet	Oligonucleotides are delivered with an Oligonucleotide Technical Data Sheet, which includes oligonucleotide name, sequence, concentration, quantity in OD and nmols, Tm, MW, size, extinction coefficient and purification data.
RP-HPLC profile	Oligonucleotides are delivered with an RP-HPLC profile for each LNA fluorescent probe
Services available upon request	• Custom design service • Aliquoting Note: Additional services may increase turn-around time.
Pricing	Please contact your local Sigma Custom Products representative
Ordering	On-line,by email or fax