Team:JMU Wuerzburg/Experiments
Dry Lab
The general Plasmodium primer-probe pair was created as described below.
To distinguish suitable sequences for primer/probe design we created a FASTA file with all available mitochondrial nucleotide sequences of Plasmodium falciparum, P. malariae, P. knowlesi, P. ovale and P. vivax from the NCBI database5. These 2080 Sequences were aligned with ClustalO1. For this step we used ClustalO, running on a computing cluster and the following bash command:
Afterwards, we created a frequency matrix from the resulting MSF file. The frequency matrix was created with Emboss Prophecy 2. We used the following parameters:
Subsequently, we calculated a new matrix based on the frequency matrix. Table 1 shows how we calculated this matrix.
Table 1: We calculated a matrix
with eight columns and as many rows as positions in the frequency matrix. In the columns,
B - F are the nucleotides Adenin (A), Cytosin (C), Guanin (G) and Thymin (T).
The first row and column are only shown for a better orientation in the matrix.
In this matrix, we searched for positions with a high value in column I
(near or equal 1) and a nucleotide summation as high as possible.
The sequence of Plasmodium is often Thymin/Adenin rich,
so we had also to look for a high Guanin/Cytosin-content.
If a suitable sequence is long enough for a primer and probe design,
we designed primers and a probe with OligoArchitectTM3.
Afterwards, we calculated the secondary structure of the primers and the probe with RNAstructure4.
Therefore we used the following parameters:
If the calculated secondary structure is moderate or low, we started testing the primers and the probe in the wet lab.
Our primer/probe pair for P. falciparum was designed as written below. We created five alignments with ClustalO1. One alignment is for P. falciparum with 1012 mitochondrial sequences of P. falciparum from NCBI and four alignments are for P. malariae, P. knowlesi, P. ovale and P. vivax5. These four alignments were calculated with mitochondrial nucleotide sequences from NCBI of each Plasmodium species5. The alignments were calculated with ClustalO with the following bash command:
We used the resulting consensus sequences and aligned these five sequences with ClustalO. Subsequently, we searched in the resulting MSF file after differences between the consensus sequence of P. falciparum and the other four consensus sequences. Afterwards, we created a frequency matrix as described in the part for Plasmodium in general and checked if our regions of interest are highly conserved as described in the part of Plasmodium in general. The primer and probe design were also carried out as described in the upper part for Plasmodium.
Furthermore, we aligned over 40 000 nucleotide sequences from NCBI5, DDBJ6 and Embl7 of Norovirus with ClustalO. We searched in the resulting alignment for highly conserved regions, as described in the part for Plasmodium in general and designed a primer/probe pair for Norovirus.
2 Rice P., Longden I. and Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics. 2000 16(6):276-277
3 https://www.sigmaaldrich.com/technical-documents/articles/biology/oligoarchitect-online.html
4 J.S. Reuter and D.H. Mathews. "RNAstructure: software for RNA secondary structure prediction and analysis." BMC Bioinformatics, 11:129. (2010).
5 https://www.ncbi.nlm.nih.gov/
6 https://www.ddbj.nig.ac.jp/index-e.html
7 https://www.ebi.ac.uk/ena
To distinguish suitable sequences for primer/probe design we created a FASTA file with all available mitochondrial nucleotide sequences of Plasmodium falciparum, P. malariae, P. knowlesi, P. ovale and P. vivax from the NCBI database5. These 2080 Sequences were aligned with ClustalO1. For this step we used ClustalO, running on a computing cluster and the following bash command:
nohup clustalo --outfmt=msf -o outputname.msf -i inputname.fasta & > nohup.log
Afterwards, we created a frequency matrix from the resulting MSF file. The frequency matrix was created with Emboss Prophecy 2. We used the following parameters:
EMBOSS data file: EDNAFULL
Threshold reporting percentage: 75
Gap opening penalty: 3.0
Gap extension penalty: 0.3
Subsequently, we calculated a new matrix based on the frequency matrix. Table 1 shows how we calculated this matrix.
Table 1: We calculated a matrix
with eight columns and as many rows as positions in the frequency matrix. In the columns,
B - F are the nucleotides Adenin (A), Cytosin (C), Guanin (G) and Thymin (T).
The first row and column are only shown for a better orientation in the matrix.
Temperature [K]: 310.15
Maximum Loop Size: 30
Maximum % Energy Difference (MFE, MEA): 10
Maximum Number of Structures (MFE, MEA): 20
Window Size (MFE, MEA): 3
Gamma (MEA): 1
Iterations (Pseudoknot Prediction): 1
Minimum Helix Length (Pseudoknot Prediction): 3
If the calculated secondary structure is moderate or low, we started testing the primers and the probe in the wet lab.
Our primer/probe pair for P. falciparum was designed as written below. We created five alignments with ClustalO1. One alignment is for P. falciparum with 1012 mitochondrial sequences of P. falciparum from NCBI and four alignments are for P. malariae, P. knowlesi, P. ovale and P. vivax5. These four alignments were calculated with mitochondrial nucleotide sequences from NCBI of each Plasmodium species5. The alignments were calculated with ClustalO with the following bash command:
nohup clustalo --outfmt=msf -o outputname.msf -i inputname.fasta & > nohup.log
We used the resulting consensus sequences and aligned these five sequences with ClustalO. Subsequently, we searched in the resulting MSF file after differences between the consensus sequence of P. falciparum and the other four consensus sequences. Afterwards, we created a frequency matrix as described in the part for Plasmodium in general and checked if our regions of interest are highly conserved as described in the part of Plasmodium in general. The primer and probe design were also carried out as described in the upper part for Plasmodium.
Furthermore, we aligned over 40 000 nucleotide sequences from NCBI5, DDBJ6 and Embl7 of Norovirus with ClustalO. We searched in the resulting alignment for highly conserved regions, as described in the part for Plasmodium in general and designed a primer/probe pair for Norovirus.
List of References
1 Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7:539 doi:10.1038/msb.2011.752 Rice P., Longden I. and Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics. 2000 16(6):276-277
3 https://www.sigmaaldrich.com/technical-documents/articles/biology/oligoarchitect-online.html
4 J.S. Reuter and D.H. Mathews. "RNAstructure: software for RNA secondary structure prediction and analysis." BMC Bioinformatics, 11:129. (2010).
5 https://www.ncbi.nlm.nih.gov/
6 https://www.ddbj.nig.ac.jp/index-e.html
7 https://www.ebi.ac.uk/ena
Hardware
Having a qPCR test system is fine. But it is only useful, if you have the specific hardware device for it. Our cycler is constructed very simply: a drawer for the test itself has two clamps to fixate the tube, so it won’t move. Underneath the drawer-slot are the operators for our test system. These operators are heat-elements, thermo sensors and relapsing magnets. To get the blood from one chamber of the tube to the next, we use a pump-lever. This pump-lever is montaged above the test-tube and runs by a small stepper-motor. The lever drives on a gear shaft that rotates by the stepper motor. At the end of this lever there is a roller, which pulls the liquid forward. At the end of the device there are LED-diodes and photocells wired to an ARDUINO computer. The diodes radiate light with 495 nm wavelength at the fluorescent probe. The photocells measure the emitted light from the probe and give the data to the ARDUINO computer. This data makes it possible to derive the presence of Plasmodium types.
Scheme of our hardware device.
Scheme of our hardware device.
Oligonucleotides
We designed the following oligonucleotieds as either Primer, Probe or our BioBrick:
Protocols
We used the following kits and protocols to conduct our experiments:
One Step RT qPCR Probe ROX L Kit (highQu)2
Quanti Nova Probe PCR Kit (Quiagen)3
LightCycler ® 480 RNA Master Hydrolysis Probes (Roche)4
LightCycler® EvoScript RNA SYBR® Green I Master (Roche)6
LightCycler® 480 SYBR Green I Master (Roche)7
innuPREP Gel Extraction Kit (Analytic Jena)9
GenElute Plasmid DNA Midiprep Kit (Analytic Jena)10
E.Z.N.A. Total RNA Kit I (Omega)11
Voltage: 100 V
Put 15 ng of the ligation mixture to 50 µl of competent cells
Place the mixture 10 minutes on ice.
Heat shock at 42°C for 45 seconds
Add 1 ml LB-Medium
Place tube at 37°C for 60 minutes and shake 300 rpm.
Centrifuge 2 min at 11000 rpm
Take 850 µl supernatant und resuspend the 200 µl.
Spread it onto the plates.
Incubate overnight at 37°C
2. Trypsinize cells with 2 ml ATV
3. Check cell-detachment under microscope
4. Add 2 ml FCS-medium to stop trypsinization
5. Resuspend cells by pipetting up and down
6. Transfer 3 ml to fresh falcon tube
7. Add 9 ml medium to the flask
8. Transfer 10 µl to the Neubauer chamber,
Count cells in a four to four square (Count 16 mini squares);
we counted 182 cells, so we had 1,82 * 10 ^6 cells per ml;
seed 1,5 * 10^5 cells per well in a 6-well plate, 1,5 * 10^5 / 1,82 * 10^6 = 82,4 µl
9. Dilute cells;
We had 6 ml in a falcon, so we add 6 ml medium
10. Prepare 2* 917,5 µl = 1,835 ml of cell-culture medium per well in a 6-well plate
11. Add 165 µl cell suspension to each well
12. Shake 6-well plate (3 pieces)
1. 0,3 - 0,5 µg GFP
2. 2,5 - 3,5 µg PUC
3. 100µl DMEM
4. Mix
5. 6 - 9 µl PEI
5. Mix
6. Incubate 15 min at room temperature (RT)
7. Drop briefly at the cells
(RT)qPCR Probe-Kis
Biozym Probe qPCR Kit (Biozym)1One Step RT qPCR Probe ROX L Kit (highQu)2
Quanti Nova Probe PCR Kit (Quiagen)3
LightCycler ® 480 RNA Master Hydrolysis Probes (Roche)4
(RT)qPCR Sybr Green Kits
LightCycler® FastStart DNA Master SYBR Green I (Roche)5LightCycler® EvoScript RNA SYBR® Green I Master (Roche)6
LightCycler® 480 SYBR Green I Master (Roche)7
DNA/Plasmid Extaction Kit
innuPREP Plasmid Mini Kit (Analytic Jena)8innuPREP Gel Extraction Kit (Analytic Jena)9
GenElute Plasmid DNA Midiprep Kit (Analytic Jena)10
E.Z.N.A. Total RNA Kit I (Omega)11
Agarose gel
1,8 % (0,9 g Agarose, 50 ml TAE buffer), 2 µl EthidiumbromidVoltage: 100 V
NEB® PCR Cloning Kit (NEB)12
cDNA Synthese with M-MuLV RT Quick Protocol (NEB)13
Template RNA 4 µl, Primer 1 µl, 10x M-MulV buffer 2 µl, M-MulVRT 1 µl, 10 mM dNTP 1 µl, H2O 11 µl 1h incubation at 42 °C
Digest with EcoRI-HF and PSTI-HF14 15
DNA 1 µg, CutSmart 10 x buffer 10 µl, EcoRI-HF 2 µl, PSTI-HF 2 µl, H2O to 100 µlLigation with T4 DNA ligase16
Agarplates with chloramphenicol resistance
10 g tryptone, 5 g yeast extract, 5 g NaCl, 10 g Agar, H2O to 1 l; autoclave; 10 mg/ml ChloramphenicolTransformation (Changed from NEB)17
Thaw chemical competent cells on icePut 15 ng of the ligation mixture to 50 µl of competent cells
Place the mixture 10 minutes on ice.
Heat shock at 42°C for 45 seconds
Add 1 ml LB-Medium
Place tube at 37°C for 60 minutes and shake 300 rpm.
Centrifuge 2 min at 11000 rpm
Take 850 µl supernatant und resuspend the 200 µl.
Spread it onto the plates.
Incubate overnight at 37°C
Sequencing18
1 µl DNA, 1 µl BigDye 1.1, 1 µl Primer (10 µM), 2 µl H2O
Thermocycler program:
1. denaturation 96°C 1min
2. denaturation 96°C 20 s
3. annealing 50°C 15 s
4. polymerisation 60°C 4 min
5. cooling 4°C ∞
Seed cells
1. Aspirate medium from cell-culture (two 75 cm2)2. Trypsinize cells with 2 ml ATV
3. Check cell-detachment under microscope
4. Add 2 ml FCS-medium to stop trypsinization
5. Resuspend cells by pipetting up and down
6. Transfer 3 ml to fresh falcon tube
7. Add 9 ml medium to the flask
8. Transfer 10 µl to the Neubauer chamber,
Count cells in a four to four square (Count 16 mini squares);
we counted 182 cells, so we had 1,82 * 10 ^6 cells per ml;
seed 1,5 * 10^5 cells per well in a 6-well plate, 1,5 * 10^5 / 1,82 * 10^6 = 82,4 µl
9. Dilute cells;
We had 6 ml in a falcon, so we add 6 ml medium
10. Prepare 2* 917,5 µl = 1,835 ml of cell-culture medium per well in a 6-well plate
11. Add 165 µl cell suspension to each well
12. Shake 6-well plate (3 pieces)
Transfection with PEI
For each well of a 6-well plate1. 0,3 - 0,5 µg GFP
2. 2,5 - 3,5 µg PUC
3. 100µl DMEM
4. Mix
5. 6 - 9 µl PEI
5. Mix
6. Incubate 15 min at room temperature (RT)
7. Drop briefly at the cells
List of References
1 https://www.biozym.com/desktopmodules/webshop/downloads/331455_170220_Biozym-Probe-qPCR-Kit.pdf2 https://www.highqu.com/1step-rt-qpcr-probe-rox-l-kit.html
3 https://www.qiagen.com/us/resources/resourcedetail?id=5167d782-9fef-4202-bc79-95f358be7d8c&lang=en
4 https://lifescience.roche.com/en_de/products/lightcycler14301-480-rna-master-hydrolysis-probes.html#documents
5 https://lifescience.roche.com/en_de/products/lightcycler-faststart-dna-master-sybr-green-i.html#documents
6 https://www.lifescience.roche.com/en_de/products/lightcycler-evoscript-rna-sybr-green-i-master.html#documents
7 https://biochimie.umontreal.ca/wp-content/uploads/sites/37/2015/11/LC480SYBRMasterguide.pdf
8 https://www.analytik-jena.de/fileadmin/content/pdf_life_science/Manual/Manual_innuPREP_Plasmid_Mini_Kit_2.0.pdf
9 https://www.analytik-jena.de/fileadmin/content/pdf_life_science/Manual/Manual_innuPREP_Gel_Extraction_Kit.pdf
10 https://www.sigmaaldrich.com/catalog/product/sigma/pld35?lang=de®ion=DE
11 http://omegabiotek.com/store/wp-content/uploads/2013/04/R6834-Total-RNA-Mini-Kit-I-Combo-Online.pdf
12 https://international.neb.com/-/media/catalog/datacards-or-manuals/manuale1202.pdf
13 https://international.neb.com/protocols/2016/04/26/first-strand-cdna-synthesis-quick-protocol-neb-m0253
14 https://international.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
15 https://nebcloner.neb.com/#!/redigest
16 https://international.neb.com/Protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202
17 https://international.neb.com/protocols/2012/05/21/transformation-protocol
18 http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_041330.pdf