Probes design

FastPCR allows the design of highly efficient probes for TaqMan (is the same as primer design) and Molecular Beacons assays as for other short or long single oligonucleotides probes (experimentally tested).
The TaqMan and Molecular Beacons assays both use a reporter and a quencher dye attached to the probe. The oligonucleotide probe hybridizes to the target sequence, and the reporter and quencher are separated either by the exonuclease activity of the Taq polymerase (5’-to-3’ polymerisation-dependent exonuclease replacement activity) for TaqMan, or because of conformation changes upon hybridization to the target sequence for the Molecular Beacons.

TaqMan Design

TaqMan probes provide an indirect measure of hybridization, as the signal continues to be generated once the reporter and quencher are separated by the exonuclease activity of the polymerase.
TaqMan probes must be designed (if possible) with a GC-content of 45-65%, a high complexity, no dimer with primers, a high Tm (60-65°C) and a probe length of 18 to 30 bp and probe Tm should be 8-10°C higher than the primers. Probes are covalently conjugated with a fluorescent reporter dye (generally the 6-carboxy-fluorescein [FAM] (Fluorescein based reporter dyes (FAM, JOE, Cal Gold, Vic, etc.) should not be conjugated to a G residue); excitation 494 nm, emission 518 nm) and a non fluorescent quencher dye (Black Hole Quencher [BHQ-1], 4'-(2-Nitro-4-toluyldiazo)-2'-methoxy-5'-methyl-azobenzene-4''-(N-ethyl)-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)) at the most 5' and most 3' base, respectively.

The BHQ family of quenchers was developed to provide excellent spectral overlapping over the entire range of commonly used reporter dyes. They efficiently quench across the electromagnetic spectrum from 480 nm into the near infrared IR-making it possible to use reporter dyes that emit anywhere within this range.

TaqMan Assay for genotyping single nucleotide polymorphisms (SNPs)

  • Probes for both alleles have a Tm of 60° to 65°C, and the difference between the Tm values of the two probes should be <1°C.
  • Probes are designed to anneal to the same strand, i.e., they should not be complementary to each other.
  • Probes do not have a G at the 5'-end, because this nucleotide interferes with fluorescence from the reporter dye that will be attached to this end.
  • Probes are designed using the strand that has fewer Gs than Cs. As far as possible, a run of ≥4 Gs is avoided.
  • The SNP is located in the middle third of the probe.
  • Probes are between 12 and 30 nt in length, although this will be determined by the Tm requirement above. In the authors’ experience, shorter probes show better discrimination than longer ones.

Molecular Beacon Design

Molecular Beacons are oligonucleotide probes that can report the presence of specific nucleic acids in homogenous solutions. They are useful in situations where it is not either possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes, such as in real-time monitoring of polymerase chain reactions in sealed tubes, or in detection assay of RNAs within living cells. Molecular Beacons are hairpin or self-dimer shaped molecules (see below) with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid. They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence.
A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence.

Molecular Beacon example sequence:

6-FAM 5'-CGCTGCtgtcgcgaccttatgagaacgcGCAGCG-3' BHQ1

 The underlined sequence at the 5' and 3' ends identifies the arm sequences that are complementary. The length of the probe sequence (18-30 nt) is chosen in such a way that the probe target hybrid is stable in the conditions of the assay. The stem sequence (5-8 nt) is chosen to ensure that the two arms hybridize to each other but not to the probe sequence (see Fig).

Design and properties of LUX (fluorogenic) PCR primers

LUX™ (Light Upon eXtension) Primers are an easy to use, highly sensitive, and efficient method for performing real-time quantitative PCR (qPCR) and RT-PCR (qRT-PCR). Each primer pair in the LUX™ system includes a fluorogenic primer with a fluorophore attached to its 3'end and a corresponding unlabeled primer. The fluorogenic primer has a short sequence tail of 6–8 nucleotides on the 5'end that is complementary to the 3'end of the primer (see Fig). The resulting hairpin secondary structure provides optimal quenching of the fluorophore. When the primer is incorporated into double-stranded DNA during PCR, the fluorophore is de-quenched and the signal increases by up to 10-fold.

Fig. LUX-primer (also oligonucleotide probes “Molecular Beacon”) formed a single molecule hairpin or alternative and stable self-dimer from two molecules.

Fluorogenic primers were designed to increase their fluorescence intensity when incorporated into the double-stranded PCR product. This design is based on a previous study that demonstrated the effects of the primary and secondary structure of oligonucleotides on the emission properties of the conjugated fluorophores. Briefly, the design factors are the presence of either a C or G as the terminal 3' nucleotide of the primer, the fluorophore being attached to the second or third base (T) from the 3' end, the presence of one or more Gs within the 3 nt flanking the labeled nucleotide and, for hairpin primers, the existence of a 5' tail that is complementary to the 3' end of the primer. The 5' tail forms a blunt-end hairpin at temperatures below its melting point. The stem of the hairpin primers have a dG ranging from –1.6 to –5.8 kcal/mol.

Example: SSR target find and design PCR LUX primers for it. Software automatically finds SSR loci and design around them PCR primers (200 bases for Forward (LUX primers) and right side from SSR loci):

> -ssr/200 –Fluxpd