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.
SECTION I: Design criteria for adjacent hybridisation probes
General guidelines
- The selection of primers can be made using the search-launcher programme
Primer
Selection or for account holders of trishul.sci.gu.edu.au using the
search-launcher command and following the instructions.
- The melting temperatures (Tm's) of the oligonucleotides are estimated on the
basis of thermodynamic data of nearest-neighbour interaction calculations using
Tm determination.
- The propensity of oligonucleotides to anneal to themselves or to other
oligonucleotides is predicted by the Oligo Selection Programme (OSP).
- For estimation of specificity, the recommended oligonucleotides are checked
against the GenBank nucleotide sequence database using the FASTA program at
Blast
- Mispairs between the hybridisation probe and non-target sequences are
placed near the centre of the probe to maximise the specificity.
Specific guidelines recommended by Idaho Technology, for designing
fluorogenic adjacent hybridisation probes
- Probe Tm's should be near equal and 5 to 10oC greater than
primer Tm's.
- The 3' end of the upstream probe should be labelled with fluorescein,
which serves as the donor in the FRET and blocks the extension of the probe.
- The 5' end of the downstream probe should be labelled with Cy5, which
which serves as the acceptor in the FRET, and the 3' end of the probe should
be phosphorylated to block the extension.
- The two probes should be separated from each other by 1 base (upto 5 bases
maybe okay).
SECTION II: Synthesis of fluorogenic probes & sample preparation
PCR Buffer Composition for 10u
| Components
| Amounts
|
| Genomic DNA | 2 ng |
|
| Forward Primer | 0.5um |
|
| Reverse Primer | 0.5um |
|
| Fluorescein-labelled probe | 0.2um |
|
| Cy-5-labelled probe | 0.2um |
|
| 10x buffer (30mM MgCl2, Idaho part# 1770* | 1ul
|
|
| dNTP | 200uM |
|
| 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:
- 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 been 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: Programing 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 F2/F1 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 45 and type of
programme set to regular, display mode set to F2/F1 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 94C for 0 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:(optional) Number of cycles 1,
type of programme is melting peaks, fluorescence acquisition time and display
mode F2/F1. Subsequent to these settings, a 2 step programme is added.
Step 1 is 45C for 20 secs with a temperature transition rate of 20C / sec.
The acquisition mode is set to none. Step 2 is 94C for 0 mins and temperature
transition rate is set to 0.1C / sec (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 F2/F1 value should
be around 2. The right hand side window shows the temperature profile and
the cycle time.
SECTION VI:
This is an example of the detection and quantitation of
Leptospira interrogans
using the fluoroscent adjacent probes method described above
SECTION I: Design criteria for adjacent hybridisation probes
General guidelines
- The selection of primers can be made using the search-launcher programme
Primer
Selection or for account holders of trishul.sci.gu.edu.au using the
search-launcher command and following the instructions.
- The melting temperatures (Tm's) of the oligonucleotides is estimated on the
basis of thermodynamic data of nearest-neighbour interaction calculations using
Tm determination
- The propensity of oligonucleotides to anneal to themselves or to other
oligonucleotides is predicted by the Oligo Selection Program (OSP).
- For estimation of specificity, the recommended oligonucleotides are checked
against the GenBank nucleotide sequence database using the FASTA program at
Blast
- Mispairs between the hybridisation probe and non-target sequences are
placed near the centre of the probe to maximise the specificity.
Specific guidelines for designing TaqMan fluorogenic probe for 5' nuclease assay
are listed in a technical bulletin of Perkin Elmer:
- Probes should be 20 to 30 bases long to ensure good hybridisation and
specificity of binding. If possible, GC content should be 40 to 60 percent.
Probe Tm, estimated by the nearest-neighbour method, should be at least 5(C
higher than the anneal/extend temperature.
- Avoid secondary structure in primers, probes, and target strands to which
the probes bind.
- Probes should neither hybridise nor overlap with the forward and reverse
primers.
- Avoid probes with long runs of a single base (that is, more than 3 or 4,
(especially G).
- Avoid placing G at the 5' end of the probe.
- When choosing between a given probe sequence and its complement, pick the
strand that gives the probe more Cs than Gs.
- Probes should be labelled with FAM as reporter dye at the 5' end, and with
TAMAA as quencher dye at the 3' end.
SECTION II: Synthesis of fluorogenic probes & sample preparation
PCR Buffer Composition for 10ul
| Components
| Amounts
|
| Genomic DNA | 2 ng |
|
| Forward Primer | 0.2um |
|
| Reverse Primer | 0.2um |
|
| TaqMan probe | 0.3um |
|
| 10x buffer (30mM MgCl2, Idaho part# 1770* | 1ul
|
|
| dNTP | 250uM |
|
| Taqpolymerase, Promega | 0.4U |
|
*1. The components can also be prepared by adding ...........
(see Mark)about receipe).
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: Programing 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).
- The settings for TaqMan hydrolysis probes is very similar to that for the
adjacent hybridization probes with two exceptions. The two programmes,
namely Denaturation programme and the Cycle programme, are used but not the
third melting curve optional programme. In addition, the fluorescence display
modeis set to F1/1 as shown below.
- 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
not set for acquisition). 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 94C 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.
- 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: Detection and quantitation of Leptospira interrogans
using fluroscent adjacent probes in a LightCycler
FIGURES FROM TONY IN COLOR
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 DNA | 2 ng |
|
| Forward Primer | 0.5um |
|
| Reverse Primer | 0.5um |
|
| SYBR Green I (1:1,000 dilution in TE) | 0.1ul |
|
| 10x buffer (30mM MgCl2, Idaho part# 1770* | 1ul
|
|
| dNTP | 200uM |
|
| 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.
- 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.
- 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).
- 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).
- 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.
- 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).
- 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)
- 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).
- 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
bharat@trishul.sci.gu.edu.au
Created: 8 June 1997
Modified: 14 Dec 1997