Real-Time PCR

LightCycler® Instruments

Roche Molecular Diagnostics has introduced the LightCycler® Instruments, the fastest thermocyclers with online monitoring of PCR based on fluorescence detection. The capillary based system works with hot and cold air allowing rapid ramping - 30 cycles can be performed within 20 minutes. The PCR process is monitored by fluorescence quantification of DNA-binding dyes for general detection of double stranded DNA, or, with hybridization probes, to monitor the amount of a specific target sequence. Some of the major advantages of online-PCR are the direct quantification of the amplicons and a minimal carry over risk with PCR products, by avoiding post-PCR handling.

Detection Channels

The probes are irradiated with a blue light–emitting diode (470nm), which excites yellow dyes including fluorescein (FAM, FITC) and SybrGreen. The emitted fluorescent light is detected in three to six channels, depending on the instrument type.

Experimental formats LightCycler® 1.x Instrument:

DNA-binding dyes

HybProbes

Hydrolysis Probes

Molecular Beacons

SYBR Green binds to double stranded DNA PCR product and its fluorescence is greatly enhanced

HybProbes hybridize on the PCR product and emit a fluorescence signal (FRET)

Hydrolysis probes contain two dyes. The reporter dye is released by hydrolysis during PCR amplification (TaqMan Format)

Molecular beacons unfold during binding to the PCR product separating the quencher, thus enhancing the fluorescence

channel 1 = 530 nm

channel 2 / 3

channel 1

channel 1

not sequence-specific

sequence-specific

sequence-specific

sequence-specific

quantification enabled

quantification enabled

quantification enabled

quantification enabled

primer-dimer can be excluded with a melting curve

mutation detection possible with melting curve

 

mutation detection possible

Experimental formats LightCycler® 2.0 Instrument:

DNA-binding dyes

HybProbes

Hydrolysis Probes

Molecular Beacons

SYBR Green binds to double stranded DNA PCR product and its fluorescence is greatly enhanced

HybProbes hybridize on the PCR product and emit a fluorescence signal (FRET)

Hydrolysis probes contain a dye and a quencher. The reporter dye is released by hydrolysis during PCR amplification (TaqMan Format)

Molecular beacons unfold during binding to the PCR product separating the quencher from the reporter dye, thus enhancing the fluorescence

channel 1 = 530 nm

channel 3, 4, 5 & 6

channel 1 & 2

channel 1 & 2

not sequence-specific

sequence-specific

sequence-specific

sequence-specific

quantification enabled

quantification enabled

quantification enabled

quantification enabled

primer-dimer can be excluded with a melting curve

mutation detection possible with melting curve

mutation detection possible

Hybridization Probe Assay

This sequence-specific fluorescence approach is based on Fluorescence Resonance Energy Transfer (FRET). During FRET, a donor fluorophore, which is excited by an LED light source, transfers its energy to an acceptor fluorophore only when positioned in the direct vicinity of the former. The acceptor fluorophore emits light of a longer wavelength, which is detected in specific channels. The LED cannot excite the acceptor dye.

HybProbes

The probes for the LightCycler® Instruments consist of a pair of oligonucleotides that can bind neighbored on a target nucleic acid. The FRET principle is dependent on the close proximity of the dyes. As long as no specific DNA is present, the energy transfer cannot take place. The amount of hybridized probes increases with the growing PCR product. The signal is proportional to the amount of amplicon.

A set of HybProbes consists of a pair of oligonucleotide probes that can hybridize adjacent on a DNA template, one labeled with fluorescein at its 3'-end (3FL) and the other labeled at its 5'-end with LC Red dye (5LC) . The free 3'-hydroxy group will usually be blocked with a phosphate against polymerase extension.

Quantification

When using external standards of known copy numbers, the LightCycler® Instruments will enable the exact determination of template amount. When working with plasmid standards, we recommend the use of dUracil-containing preparations to minimize the contamination risk. External custom dU-standards are available from GenExpress, Berlin.

Mutation Detection

Besides quantification of template the HybProbe format also allows the detection of mutations by monitoring changes in the melting behavior of the probes.

Design of HybProbes

The probes should be located on the same strand, positioned in the middle of the amplicon or near to, but not overlapping with the primers of the opposite strand. A distance of one to five bases between both fluorophores is required to ensure optimal performance (4 to 25 Å molecular distance).

Sequence

Self-complementary, palindromic and repetitive sequences should be avoided when designing primers. In general, the sequences should have a balanced base content without local high spots of G/C bases.

Melting Point

The melting point, Tm, is the temperature at which half of the complementary molecules are hybridized. The Tm can be calculated by hand, following the 2/4°C rule but will be more accurate when calculated based on the thermo-dynamic properties of dinucleoside pairs. An algorithm for the calculation is available on our homepage ( www.TIB-MOLBIOL.com). In general, the melting point of the probes should be 5-10°C higher than the Tm of the primers.

Mutation Detection

It is important that the probe spanning the mutation has a lower Tm than its partner probe (anchor probe).

Custom Design

On request we evaluate your system, give you our suggestions or design your HybProbe assays based on your PCR primers. We work with different programs to analyze Tm, thermodynamic stability, hybridization kinetics, sequence structure, false priming and Genbank sequences.

HybProbes are available in the following product amounts: 1 nmol, 3 nmol and 30 nmol. Other scales are available upon request. The probe prices include the synthesis of the corresponding oligonucleotides (max. 30 bases each). All products are purified by a multistep HPLC procedure and final desalting. Delivery lyophilized.

Disclaimer:
TIB MOLBIOL offers the custom synthesis of HybProbes for the LightCycler®™ under license of Roche Diagnostics GmbH. TIB MOLBIOL has LightCycler® systems located in their various production facilities and welcomes cooperation projects.

Literature:

  • Wittwer et al. BioTechniques (1997) 22, 130-138
  • Wittwer et al. BioTechniques (1997) 22, 176-181

Addresses:

Berlin, Freehold, Genoa, Poznan, Barcelona

Code               

Product

Amout

17-0640-03

Custom Synthesis 5'-LC640, 3'-phosphate

3 nmol

17-0640-06

Custom Synthesis 5'-LC640, 3'-phosphate

2 x 3 nmol

17-0640-30

Custom Synthesis 5'-LC640, 3'-phosphate

30 nmol

17-0640-33

Custom Synthesis 5'-LC640, 3'-phosphate

10 x 3 nmol

17-0644-03

Custom Synthesis int. dT-C2-LC640, 3'-OH

3 nmol

17-0644-30

Custom Synthesis int. dT-C2-LC640, 3'-OH

30 nmol

17-2121-03

Custom Synthesis 3'-fluorescein

3 nmol

17-2121-06

Custom Synthesis 3'-fluorescein

2 x 3 nmol

17-2121-30

Custom Synthesis 3'-fluorescein

30 nmol

17-2121-33

Custom Synthesis 3'-fluorescein

10 x 3 nmol

17-2124-03

Custom Synthesis int. dT-C2-fluorescein

3 nmol

17-2124-30

Custom Synthesis int. dT-C2-fluorescein

30 nmol

17-2640-01

Custom Synthesis HybProbe 640 Set 5LC /3FL

1 nmol

17-2640-03

Custom Synthesis HybProbe 640 Set 5LC /3FL

3 nmol

17-2640-06

Custom Synthesis HybProbe 640 Set 5LC /3FL

2 x 3 nmol

17-2640-30

Custom Synthesis HybProbe 640 Set 5LC /3FL

30 nmol

17-2640-33

Custom Synthesis HybProbe 640 Set 5LC /3FL

10 x 3 nmol

Disclaimer:
Custom synthesis of hybridization probes and oligonucleotides containing LightCycler® Red dyes under license of Roche Diagnostics GmbH. All products are for research use only. Not for use in diagnostic procedures. No resale. LightCycler® is a trademark of Roche. The use of the LightCycler® logo with permission of Roche Diagnostics GmbH.