Custom DNA Probes

LightCycler® Probes

LightCycler probes are highly sensitive and sequence-specific fluorescent probes designed for use with the Roche LightCycler instruments.

Choose LightCycler Probes for:

  • SNP detection
  • Allele discrimination
  • Pathogen detection
  • Multiplexing
  • Viral load quantification
  • Gene expression analysis
  • Gene copy determination

Several fluorophores are available and are suitable for multiplex analysis.

Benefits of Using LightCycler Probes include:

  • Increased specificity
  • Probe preserved during the reaction
  • Increased thermal stability and hybridization specificity
  • Greater accuracy in SNP detection and allele discrimination
  • Easier and more sensitive probe designs for problematic target sequences

Add LNA to Your Probe for:

  • Increased thermal stability and hybridization specificity
  • Greater accuracy in SNP detection and allele discrimination
  • Easier and more sensitive probe designs for problematic target sequences

How LightCycler Probes Work

A LightCycler Probe system consists of a pair of single-stranded fluorescent-labeled oligonucleotides. Oligo Probe 1 is labeled at the 3' end with a donor fluorophore dye and Oligo Probe 2 is labeled at its 5' end with one of a few available acceptor fluorophore dyes. The free 3' hydroxyl group of Probe 2 must be blocked with a phosphate group (P) to prevent DNA polymerase extension. There should be a spacer of 1 to 5 nt to separate the two probes from each other. The structures and mechanism of a LightCycler probe are shown below.

During the annealing step of qPCR, the PCR primers and the LightCycler Probes hybridize to their specific target regions bringing the donor dye into close proximity to the acceptor dye. When the donor dye is excited by light from the LightCycler instrument, energy is transferred by Fluorescence Resonance Energy Transfer (FRET) from the donor to the acceptor dye. The acceptor fluorophore's emission wavelength is detected. The increase in fluorescence signal is directly proportional to the amount of target DNA.

Product Features include:

  • Amounts: 0.1, 0.25, 1.5, and 15 OD
  • Purification: HPLC
  • Sequence Lengths: 15 - 40 bases
  • Quality Control: 100% mass spectrometry
  • Format: Supplied dry in amber tubes
  • Custom formats available (normalization, special plates, etc.)

Sigma's probes are provided in a format to simplify your experimental planning.

Guaranteed Yields of LightCycler Probes

Guaranteed
OD Yield		 Approx. No.
of nmoles		 Approx. No.
of µg		 Approx. No.
of Reactions*
0.1 0.4 3.2 80
0.25 1 8 200
1.5 6 48 1,200
15 60 480 12,000
*Estimate is based on 4 nmoles or 32 µg for 1 OD and 200 nM in 25 µl reaction (5.0 pmol/reaction). Estimate is based on an average sequence length of 25 bases.

The recommended constructs for LightCycler Probe 1 and LightCycler Probe 2 are listed in the tables below:

Labels and Modifications for LightCycler Probes

Probe 1
3' donor fluorophore Fluorescein
Probe 2*
3' end Phosphate
5' end LightCycler Red 610, 640, 670 and 705
*For enhanced discrimination, LNA can be incorporated into Probe 2.

Sigma is a licensed supplier of a variety of dyes and quenchers and continually adds to its portfolio of new chemistries. For assistance in the design of your LightCycler Probes and assays, use our online design tool at sigma.com/probedesignonline.

 

CleanAmp dNTPs