The RapidCycler and LightCycler are produced by Idaho Technology, USA. The RapidCycler is the worlds fastest PCR machine requiring less than 10 minutes for amplification. The LightCycler is similar to the RapidCycler but in addition is capable of performing Real-Time Dection of PCR products during amplification. If you need more information on RapidCyclers and / or LightCyclers than follow the link to the Idaho Technology homepage

Below is the research that is being conducted in my laboratory, the Molecular Microbial Biotechnology Laboratory at Griffith University using a RapidCycler and LightCyclar.

Adjacent Hybridization Probes

SECTION I: Design criteria for adjacent hybridisation probes

General guidelines

Specific guidelines recommended by Idaho Technology, for designing fluorogenic adjacent hybridisation probes

SECTION II: Synthesis of fluorogenic probes & sample preparation

PCR Buffer Composition for 10u
Components Amounts
Genomic DNA2 ng
Forward Primer0.5um
Reverse Primer0.5um
Fluorescein-labelled probe0.2um
Cy-5-labelled probe0.2um
10x buffer (30mM MgCl2, Idaho part# 1770*1ul
dNTP200uM
Taqpolymerase, Promega 0.4U
*1. The components can also be prepared by adding ........... (see Mark)about receipe).
2. It may be necessary to lower the concentration of the fluorescein-labelled probe from the recommended 0.2um to 0.16um in order to bring the initial fluorescence reading to about 8 within the 10-point scale of the LightCycler.

SECTION III: Loading samples into capillaries:

SECTION IV: Switching on and setting up the LightCycler

SECTION V: Programing the LightCycler

SECTION VI:

This is an example of the detection and quantitation of Leptospira interrogans using the fluoroscent adjacent probes method described above

TaqMan Probes

SECTION I: Design criteria for adjacent hybridisation probes

General guidelines

Specific guidelines for designing TaqMan fluorogenic probe for 5' nuclease assay are listed in a technical bulletin of Perkin Elmer:

SECTION II: Synthesis of fluorogenic probes & sample preparation

PCR Buffer Composition for 10ul
Components Amounts
Genomic DNA2 ng
Forward Primer0.2um
Reverse Primer0.2um
TaqMan probe0.3um
10x buffer (30mM MgCl2, Idaho part# 1770*1ul
dNTP250uM
Taqpolymerase, Promega 0.4U
*1. The components can also be prepared by adding ........... (see Mark)about receipe).

SECTION III: Loading samples into capillaries:

SECTION IV: Switching on and setting up the LightCycler

SECTION V: Programing the LightCycler

SECTION VI: Detection and quantitation of Leptospira interrogans using fluroscent adjacent probes in a LightCycler

FIGURES FROM TONY IN COLOR

Melting Curves of PCR products using SYBR Green I

SECTION I: Design criteria

This is a straight forward PCR technique. Flurogenic probes are not required but instead SYBR Green I is added to the PCR reaction mixture. SYBR Green if added in the right concentration will not inhibit amplification. During PCR, the dye will intercalate in the products. Fluoroscence will released as a result of strand separation resulting in a melting curve from which the Tm of the PCR product can be determined.

SECTION II: Sample preparation

A typical reaction mix consists of the following:

PCR Buffer Composition for 10u
Components Amounts
Genomic DNA2 ng
Forward Primer0.5um
Reverse Primer0.5um
SYBR Green I (1:1,000 dilution in TE) 0.1ul
10x buffer (30mM MgCl2, Idaho part# 1770*1ul
dNTP200uM
Taqpolymerase, Promega 0.4U
*1. The components can also be prepared by adding ........... (see Mark)about receipe).
The SYBR Green I dye concentrate (Molecular Probes, http://www ) is a viscous DMSO containing solution making pipetting difficult. It may be necessary to adjust the concentration emperically so that the initial baseline fluorescence is approximately 2 on the 10 point scale of the LightCycler.

SECTION III: Loading samples into capillaries:

  • Pipette 10ul sample into the special capillary tubes. Place the cap on the mouth of the capillary, and place this assembly in the rotor adaptors that have positioned in the microfuge and spin briefly

  • Remove the capillaries and snap seal the caps onto the capillaries by pushing the caps against the side of a bench in a horizontal position.

  • Dip the capillary in methanol and wipe clean gently with tissue to remove grease and oil from the surface as this may interfere with fluorescence detection.

  • You are now ready to insert the capillaries into position 1 of the LightCycler carousel. This should be done very gently to minimise breakage. If breakage occurs than refer to the LightCycler user guide on how to remove the broken glass from inside the chamber.

  • If there are less than 24 samples, then place blanks that have been provided at positions 23 and 24 as well as the last two positions after the sample capillaries. This is to stop stray light interefering with fluorescence detection.

  • Now rotate the carousel so that position of sample 1 is slightly to the right of the centre.

SECTION IV: Switching on and setting up the LightCycler

  • Switch the computer on and double click on the LightCycler icon. Check that the screen display resolution is set to 1024 x 768, 256 color. This can be done by clicking the icon on th bottom right hand corner of the screen next to the computer clock.

  • Wait for the prompt & turn on LightCycler to be displayed, then turn on the LightCycler (the switch for this is at the back of the LightCycler). Now, click the button on the display in order for the computer and the LightCycler to talk to each other.

  • Wait and another display window similar to this will appear.

  • Now click on the Lightcycler button. You are now ready to set up the programme.

SECTION V: Programming the LightCycler

  • The programme depends on the analysis you wish to undertake. The choices in the analysis are: adjacent probes (with optional melting curve acquisition which will be required only for the first run in order to find out the optimal probe hybridization temperature), TaqMan probes, SYBR green I (always with melting curve acquisition for confirming product identity).

  • Typical settings for adjacent probe hybridization includes two programmes, namely Denaturation programme and the Cycle programme. A third programme is optional and perhaps is useful for the first couple of runs in order to determine the optimal probe hybridization.

    • Denature programme: Number of cycles set to 1 and type of programme set to regular, display mode set to F1/1 and fluorescence acquistion time to 0 (but this doesnt matter as no fluorescence will is as no fluorescence is emitted). Initial deanturation which is always set at 94C for 15 secs at a temperature transition rate of 20C / sec. Acquisition mode set at none.

    • Cycle programme: Number of cycles set to 30 and type of programme set to regular, display mode set to F1/1 and fluorescence acquistion time to 100 msecs. Following this settings, multple steps can be entered but a typical 2 step cycle programme is shown as an example. Step 1: Cycle denaturation is always set at 95C for 15 secs, temperature transition rate of 20C/sec, acquisition mode set at none. Step 2: Combined annealing and extension step (but this could be separated into two steps if necessary) set at 60C for 20 secs with temperature transition rate set at 20C / sec. Acquisition mode is set to single.

    • Melting Programme: Number of cycles 2, type of programme is melting peaks, fluorescence acquisition time and display mode F1/1. Subsequent to these settings, a 2 step programme is added. step 1 is 72C for 20 secs with a temperature transition rate of 20C / sec. The acquisition mode is set to step. Step 2 is 95C for 0 sec and temperature transition rate is set to 0.2 / sec (the slowest rate.

  • Click load button and 2 superimposed display windows will pop up. In the uppermost window entitled "please name this run", type in a new file name (usually date_month_year eg 06_03_97). This file will be saved in the directory "Data". You will need to move the saved file to your own folder under director "Data" immediately after the run has finished. This will prevent the data files from being overwritten by other users who will follow the same file naming scheme.

  • Press the save button. A new window "Dialogue" will pop up. Type in the number of samples that have been loaded and press okay.

  • Click the run / stop button.

  • 3 windows will pop up. The window on the left shows the fluorescene of the probes of the sample and the F1/1 value should be around 2. The right hand side window shows the temperature profile and the cycle time.

SECTION VI: Differentiation of Leptospira biflexa, and Leptonema on the basis of melting curves and melting temperature of PCR products generated in the presence of SYBR Green I.


Publications

  1. Woo, T.H., Smythe, L.D., Symonds, M., Norris, M., Dohnt, and Patel, B.K.C. (1996). Rapid identification between Leptospira and Leptonema species. FEMS Microbiology Letters 142:85-90.

  2. Woo, T. H.S., Smythe, L.D., Symonds, M.L, Norris, M.A., Dohnt, M.F. and Patel, B.K.C. (1997). Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of the 23S ribosomal DNA. FEMS Microbiology Letters (in the press).

  3. Woo, T.H.S., Patel, B.K.C., Smythe, L.D., Symonds, M.L, Norris, M.A., and Dohnt, M.F. (1997). Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans. FEMS Microbiology Letters (in the press).

  4. Woo, T. H.S., Patel, B.K.C., L.D., Symonds, M.L, Norris, M.A., Dohnt, M.F. and Smythe, L.D. (1997). Identification of Leptospira interrogans by continuous monitoring of flurogenic adjacent hybridization probes during PCR. Journal of Clinical Microbiology 35(12):3140-3146.

  5. Woo, T.H.S., Patel, B.K.C., Cinco, M., Smythe, L.D., Symonds, M.L, Norris, M.A., and Dohnt, M.F. (1997). Identification of Leptospira biflexa by continuous monitoring of fluorescence during rapid cycle PCR. Applied and Environmental Microbiology (revision submitted).

  6. Woo, T.H.S., Patel, B.K.C., Cinco, M., Smythe, L.D., Symonds, M.L, Norris, M.A., and Dohnt, M.F. (1997). Real-time Homogeneous Assay of Rapid Cycle PCR PCR Product for the Identification of Leptonema illini. Analytical Biochemistry (in the press)

  7. Woo, T.H.S., Patel, B.K.C., Smythe, L.D., Symonds, M.L, Norris, M.A. and and Dohnt, M.F. (1997). Identification of Pathogenic Leptospira by TaqMan Probe in a LightCyclerTM. Analytical Biochemistry (in the press).

  8. Woo, T.H.S., Patel, B.K.C., Smythe, L.D., Symonds, M.L, Norris, M.A., Weyant, R.S. and Dohnt, M.F. (1998). Identification of Leptospira inadai by Continuous Monitoring of Fluorescence During Rapid Cycle PCR. Systematic and Applied Microbiology (in the press).

Send comments, errors and suggestions to B.Patel@griffith.edu.au
Created: 8 June 1997
Modified: 14 Dec 1997