Tabulación encuestas

4.2.3.1 Optimisation of PCR cycling profiles

The conditions for PCR reaction and cycling reported for the same primer set cannot be applied directly, since they usually depend on the chemicals and equipment used in the particular laboratory. Optimization of PCR involves testing different temperature conditions and cycling in order to get a specific, well separated and repeatable PCR product, which is very important for the accurate estimation of allele sizes.

Touchdown PCR is used because of tailing approach choosen to fluorescently label PCR product as described by Schuelke et al. (2000). It is used also as a strategy to increase the specificity of amplification. It runs with two steps of cycling at different annealing temperatures. The first set of cycles starts at a high annealing temperature to ensure the maximum specificity of the first primer-template hybridisation, followed by a gradual decrease in the annealing temperature to ensure specific amplification; then, in the second step, several cycles are performed at the lowest annealing temperature to provide high yields of the first product (Weising et al., 2005).

The initial PCR cycling and thermal profile used for the optimisation of the PCR products of all microsatellite loci used in this study consisted of: one cycle of 94°C for 5 minutes, 5 touchdown cycles of 94°C for 30 s, 60°C with 1.0°C lower per cycle for 45 s, 72°C for 1.30 minutes, and then 30 cycles of 94°C for 30 s, 55 °C for 45 s, 72°C for 1.30 minutes. The final elongation step was performed at 72°C for 8 minutes.

Twenty loci (DCA5, DCA15, GAPU59, GAPU71B, GAPU89, GAPU101, UDO24, EMO 90, SNB and SiBi), with some minor modifications for some of them in annealing temperature or/and number of cycles, had good amplification and an easy to score pattern with this PCR profile.

The remaining six loci, DCA3, DCA9, DCA11, DCA14, DCA16, and DCA18, tend to produce slight to medium levels of stutter bands. Stutters differ in size by 2 bp and occur due to the slippage of DNA polymerase during the extension step of amplification (Weising et al., 1995).

In our study, we made several attempts to reduce stuttering at these loci to provide a well- defined allelic pattern, such as:

1-decreasing the amount of DNA template from 20 ng to 5 ng, in order to proportionally decrease the amount of contaminants in the PCR reaction.

2-applying a short PCR (95°C for 5 min, 30 cycles of 94°C for 2 s, 55°C for 2 s, 72°c for 8s and final elongation step in 72°C for 8 min).

3-purifying of PCR product by reprecipitation with 1/5 volume 3M sodium acetate Ph=4.5 and isopropanol, followed by 95 % ethanol washing and redilution of purified PCR product in ddH2O.

4-modifying the initial touchdown PCR conditions by increasing the time of the final elongation step from 72°C for 8 minutes to 65°C for 45 min.

4.2.3.2 PCR amplification

The PCR reactions were performed in 15 µl final volume containing 20 ng genomic DNA, 5x PCR buffer (Promega), 2 mM MgCl2 (Promega), 0.2 mM of each of the dNTPs

(Sigma), 0.2 µM of each primer, 0.25 µM fluorescently labelled M13 tail and 0.3 U Taq DNA Polymerase (Promega). The forward primer was labelled with 6-FAM, VIC, NED or PET fluorescent dye. Amplification was carried out in a GeneAmp 9600 thermal cycler (Applied Biosystem) using the temperature profiles given in Table 4 for each of the primers.

Table 4: Differences in PCR conditions applied for each microsatellite Preglednica 4: Razlike v PCR pogojih za posamezen mikrosatelit

Microsatellite primer Touchdown T in C°/ Cycles / Final elongation step

GAPU59, GAPU89, GAPU71B,

GAPU101, UDO24, EMO90, DCA15 Touchdown cycles: 60°C -55°C (-1.0) / 30 cycles

Final elongation: 72 °C-8 min

DCA09, DCA16, DCA18 Touchdown cycles :60°C -55°C (-1.0) / 30 cycle

Final elongation: 65 °C-45 min

DCA05 Touchdown cycles: 62°C -57°C (-1.0) / 25 cycle

Final elongation: 72 °C-8 min

DCA03, DCA11, DCA14 Touchdown cycles: 60°C -55°C (-1.0) /30 cycle

Final elongation: 65 °C-45 min SiBi03, SiBi04, SiBi05, SiBi07, SiBi11,

SiBi19

Touchdown cycles : 58°C -53°C (-1.0)/ 30 cycle Final elongation: 72 °C-8 min

SNB03, SNB11, SNB14, SNB19, SNB20

Touchdown cycles: 60°C -55°C (-1.0) /30 cycle Final elongation: 72 °C-8 min

SNB 22

Touchdown cycles: 60°C -55°C (-1.0) / 35 cycle Final elongation: 72 °C-8 min

4.2.3.3 Electrophoretic analysis and fragment detection

The separation of amplified fragments was carried out with capillary electrophoresis using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems), which relies on automated laser detection of fluorescently labelled DNA fragments.

To reduce the time and cost of PCR characterization, we used the fluorescent labelling method described by Schuelke, (2000). This method consists of the addition at the 5’ end of the forward primer of a specific sequence (M13 tail) of 18 bp long (5'- TGTAAAACGACGGCCAGT-3') labelled with one of the fluorescent dyes 6-FAM, VIC, NED and PET. Different fluorescent labelling of PCR products enables analysis of four loci in the same capillary injection. Fragment scoring is based on different dyes of amplified loci, reducing the time and costs of the analysis. Four µl of four PCR products differently labelled were mixed together (4µl each). Hi-Di formamide (10.6 μl of) and size standard GeneScan-600 LIZ (0.5 μl) (Applied Biosystems) were mixed with 1 µl of the merged PCR product and then separated by capillary electrophoresis.

The sizes of alleles were determined against the internal standart GeneScan 600 LIZ applied with the samples, while allele naming was done using GeneMapper software version 4.0 (Applied Biosystems). Genotypes were manually scored to avoid errors attributed to the automated sizing, which usually increase in the case of stutters. Particular attention was paid during the size determination of fragments to potential genotyping error factors, such as allele dropout, which can lead to a decrease in sample heterozygosity, andstuttering patterns, which can hide the true allele peak. The resulting low frequency alleles occurring ≤ 5 times were double checked on the original pherogram and any genotyping errors were corrected accordingly. Genotypes showing only one allele were considered homozygous for the analysed locus.