2.3 Molecular biology methods
2.3.4 Polymerase Chain Reaction (PCR)
For testing the cDNA quality, semi-quantitative RT-PCR and genotyping of plants and bacteria KAPA2G Fast 2x ReadyMix with Dye (Peqlab, Erlangen, Germany) was used with reaction mix and cycling conditions as depicted in table 2.1. DNA fragments to be used in cloning procedures were amplified with iProof polymerase (Bio-Rad, Hercules, California, USA) as detailed in table 2.2. This polymerase has a 3' - 5' exonuclease activity, thereby allowing proofreading of amplified fragments.
Table 2.1 Protocol for PCR with KAPA2G Fast 2x ReadyMix with Dye
Initital denaturation was conducted for 5 min in case template was genomic DNA and for 3 min for other template DNA. Annealing temperature was approx. Tm (see equation 3, 8.2.2, supp.) of the primer with the lower Tm
(either forward or reverse primer) and adjusted as necessary. Extension time was set to 1 s if amplicon length was below 1000 bp. Above 1000 bp, 15 s / 1000 bp were used. Occasionally extension time was adjusted as necessary.
reaction mix volume [µl] components
7.5 2x Ready Mix
0.75 primer for (10 µM)
0.75 primer rev (10 µM)
as needed DNA
to 15 H2Odeion
thermo cycler
program temperature [°C] time cycles
initial denaturation 95 template specific 1
denaturation 95 15 sec
35 annealing primer specific 10 sec
extension 72 amplicon specific
Genotyping of bacteria colonies after transformation was conducted with a colony PCR. Bacterial material was taken under sterile conditions from individual colonies by tipping pipette tips gently into the colony, inserting the pipette tip into 10 µl autoclaved H2Odeion and pipetting several times up and down. The shearing forces support opening of cell membranes. Kapa2G polymerase (table 2.1) was
used for amplifying the insert with plasmid-binding primers. In order to ensure opening of cellular membranes of sufficient cells, 1 % Triton X-100 was added to the reaction mix.
For details regarding primer generation, see 8.2.1, supp..
The thermocyclers Primus 96 advanced Thermocycler (MWG-Biotech, Ebersberg, Germany) and Primus Thermocycler (MWG-Biotech, Ebersberg, Germany) were used for all PCRs.
Table 2.2 Protocol for PCR with iProofTM High-Fidelity PCR kit
Annealing temperature was approx. Tm of the primer with the lower Tm (see 8.2.2, supp.) and adjusted as necessary. Extension time was set to 30 s / 1000 bp. (iProofTM High-Fidelity PCR kit, Bio-Rad Laboratories, Inc.
Hercules, California, USA)
reaction mix volume [µl] components
10 5x iProof HF buffer
1 dNTP mix (10 mM)
2.5 primer for (10 µM)
2.5 primer rev (10 µM)
0.5 iProof polymerase
as needed DNA
to 50 H2Odeion
thermo cycler program temperature [°C] time cycles
initial denaturation 95 3 min 1
denaturation 95 15 sec
35 annealing primer specific 30 sec
extension 72 amplicon specific
2.3.4.1 Reverse Transcription (RT)-PCR cDNA synthesis
Semi-quantitative and quantitative RT-PCR is used to visualize transcript levels of the gene of interest.
Before PCR can be conducted, total RNA is transcribed into complementary DNA (cDNA) by use of the Invitrogen SuperScript VILO™ cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) as visualized in table 2.3. The kit provides an enzyme mix including the SuperScript™ III Reverse Transcriptase which generates cDNA in amounts which are linear in relation to RNA concentration. Total RNA is transcribed by use of random primers as provided by the manufacturer.
Table 2.3 cDNA synthesis protocol for the Superscript VILOTM cDNA synthase
Superscript VILOTM cDNA synthase, Thermo Fisher, Thermo Fisher Scientific, Waltham, Massachusetts, USA
reaction mix components volume [µl]
VILO reaction mix 4
enzyme mix 2
RNA as needed
H2Odeion to 20
incubation
program temperature [°C] time
25 10 min
42 60 min
85 5 min
-20 until further use
Semi-quantitative RT-PCR
From the cDNA, the gene of interest is amplified with gene-specific primers (8.2.3, supp.). The amplicon concentration increases during PCR according to a logarithmic curve. In case of semi-quantitative RT-PCR, PCR is stopped before the end of the logarithmic phase is reached. As a result, amplicon amounts are linear in relation to cDNA amounts and therefore to RNA amounts. As a result, PCR products display transcript abundance of a particular gene in specific plant organs or under defined environmental conditions. Amplicon amounts are visualized by fluorescence intensity via Ethidium Bromide after gel electrophoresis (see 2.3.5).
Quantitative RT-PCR
Quantitative RT-PCR (qRT-PCR) relies on monitoring of amplicon amounts during all times of real time PCR. For the polymerase reaction, the EvaGreen® QPCR-Mix II (ROX) (my-budget, Bio-Budget Technologies, Krefeld, Germany) was used (see table 2.4). The mix contains the EvaGreen dye and an internal reference dye (glycine conjugate of 5-carboxy-X-rhodamine, succinimidyl ester (ROX)).
EvaGreen binds to the DNA, forming a fluorescing dye-DNA complex. ROX as the internal reference dye on the other hand ensures that different fluorescence signals don't derive from different reaction mix volumes. Samples were readied in 48 well plates (MicroAmpTM Fast Optical 48 Well Reaction Plate, 0.1 ml, Applied Biosystems, Thermo Fisher Scientific Waltham, Massachusetts, USA) and well plates were covered with a plastic film (MicroAmpTM 48 Well Optical Adhesive Film, Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA) which ensures airtight covering of wells. Fluorescence intensity data were collected throughout the PCR process (table 2.4) for each sample individually by the StepOneTM Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Table 2.4 Protocol for PCR with 5x QPCR Mix EvaGreen®
5x QPCR Mix EvaGreen® my-budget, Bio-Budget Technologies, Krefeld, Germany
reaction mix volume [µl] components
4 5x Eva Green
0.1 primer for (50 µM)
0.1 primer rev (50 µM)
2 DNA (2.5 - 5 ng/µl)
to 20 H2Odeion
thermo cycler
program temperature [°C] time cycles
initial denaturation 95 10 min 1
(holding stage)
denaturation 95 15 s 40
(cycling stage)
elongation 60 1 min
denaturation 95 15 s
1 (melt curve stage)
elongation 10 1 min
temperature gradient 60 - 95 0.3 °C / min
final denaturation 95 15 s
For data analysis the comparative CT method (ΔΔ CT method with CT for cycle threshold) was employed in order to determine the relative target quantity in samples. This method uses the cycle number at which fluorescence crosses a set threshold (CT) in the amplification plot. The threshold is set in the exponential phase of the amplification curve, as automatically calculated by the StepOne™
Software (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA) depending on the baseline and the amplification curve. CT values are given for each well of the 48 well plates. For each sample, three wells are prepared as technical replicates. A housekeeping gene, for example expression of GAPDH is monitored for each investigated genotype or condition as well as for the control.
Data were interpreted according to the manufacturer's instructions with the ΔΔ CT method, employing the following equation (equation 2):
(2)
Mean CT and standard deviation was calculated for each sample from the three technical replicates.
Mean CT of the endogenous control was subtracted from mean sample CT which results in Δ CT. From Δ CT of samples of interest (investigated conditions or mutants), Δ CT of the control (control conditions or wild type) are subtracted for each gene, which gives ΔΔ CT values. In order to calculate the -fold expression of genes compared to control (fold difference in expression), equation 2 is employed.
2.3.4.2 Organ specific expression analysis
Gene expression in plant organs was investigated in plants grown under different conditions. Leaf and whole root systems were collected from plants cultivated for 4 weeks in hydroponics (2.2.2).
Investigation of gene expression specifically in different sections of the root system (3.1.2) was conducted with 5 weeks old plants grown on soil in order to obtain sufficient root material for the analysis. Root systems were quickly cleaned from soil particles under running tap water and arranged as shown in figure 2.2. As indicated by a white line in the same figure, basal and apical root parts were separated with a razor blade. The basal part entailed thickened roots which are subjected to secondary root growth. The apical section consisted of the remaining root system of fine roots.
Subsequently, samples were quickly frozen in liquid nitrogen and further prepared as explained in 2.3.2 - 2.3.4.1.
Fig. 2.2 Sample preparation for organ-specific expression analysis
Root system was separated along the white line into a basal section with mainly peridermal suberin and an apical section consisting of fine roots. The scale bar represents 1 cm.
2.3.4.3 Identification of T-DNA insertion and enhancer trap lines
T-DNA and enhancer trap lines both rely on insertion of foreign DNA into the gene of interest and thereby disrupting the native sequence. Genotyping by competitive PCR allows identification of plants either homozygous for the insertion, of wild type plants or heterozygous plants. A set of three primers was used, two of them binding in the gene and one primer binding in the T-DNA or in the enhancer trap (ET) transposable Ds element (Sundaresan et al., 1995). Looking at the gene-binding primers, the forward primer binds upstream of the insertion site, the reverse primer downstream and together they allow the amplification of a gene segment in the wild type. In the mutant, the long foreign DNA between the primers increases their distance to each other which results in an amplicon too large to amplify with standard PCR conditions. The third primer which binds in the foreign DNA is a reverse primer. It allows amplification of a fragment together with the forward primer binding in the gene. The resulting amplicon differs in length from the wild type amplicon, allowing the distinction of wild type and mutant. In case of heterozygous plants, both amplicons are generated.
basal apical