UG - Faculty of Sciences - Vakgroep Biology - Algologie en PAE
Het stage-onderwerp omvat de moleculaire verwerking van een groot experiment dat in microdammen in Ethiopië werd uitgevoerd in september 2006.
De moleculaire diversiteit van een cyanobacteriënbloei wordt bepaald via denaturerende gradient gel electroforese. A.d.h.v. PCR zullen toxine-genen gedetecteerd worden en via ELISA zal de toxine-concentratie bepaald worden. Mogelijks zal er ook gecloneerd worden en real time PCR uitgevoerd worden.
Door de student toe te passen methoden en technieken: extractie van DNA uit celculturen, PCR amplificatie van ITS2 rDNA, het scheiden van de verschillende ITSgenodemes via Denaturerende Gradient Gel Electroforese (DGGE). PCR amplificatie van de verschillende microsatelliet loci die gebruikt worden voor fragmentanalyse. Indien nodig wordt ook de basenpaarsequentie van de bekomen fragmenten bepaald.
In a lot of sample campaigns there is no possibility to immediately freeze-dry the samples for research. Freeze-drying is the golden standard for preserving biological samples. The aim of this thesis is to determine which conservation buffer at different temperatures and different time periods is the best to obtain the diversity of prokaryotes, when the samples can’t immediately be stored at -80°C.
The used samples are from the OMES project. These samples where then dived in three different conservation buffers: 96% ethanol, SLB and RNAlater. After different periods of preservation (one week, two weeks and four weeks) at different temperatures (roomtemperature, 4°C and -20°C) the DNA was extracted from the samples. From the extracted DNA, the 16S gene was amplified and then sequenced on the MinION. Storage on ethanol gives enormous variation in the composition of the samples. Even after one week in cold temperatures, the composition shows already variability compared with the standard. RNAlater gives less variations with the standard. RNAlater gives the most variations at room temperature. But at colder temperatures after a longer period, the composition has nevertheless changed compared to the standard. RNAlater protects the samples best against degradation. Storage in SLB buffer mainly varies at room temperature, with much degradation after four weeks. After four weeks it shows the most consistent results if kept on colder temperatures compared to the standard.
The conclusion is that SLB at 4°C and -20°C is the best preservation method. Even after four weeks there’s almost no difference between SLB and samples immediately stored at -80°C (which is considered as the standard). At room temperature there is a difference for all the buffers, so this is no good way to preserve the samples.
This research is a continuation of chapter five of “What’s in a name? On the importance of species delineation in Dictyotales, an order of brown algae” written by Frédérique Steen.
This research will test the genetic diversity inside and between the multiple populations. There were supposed to be more sample locations, but these did not happen due to Corona. The genetic diversity will be analysed by amplifying and analysing microsatellite sequences present in the genomic DNA. Through analysing this data, the clonality and similarity of the tested samples can be calculated. Via these two factors the genetic diversity can be interpreted.
The first step of this research is obtaining very pure genomic DNA, this DNA will be amplified by using a multiplex PCR. While the DNA sequences are being amplified a fluorescent marker is added, this marker is present in the fluorescent forward primer added during the amplification.
When the sequences are amplified they need to go through quality control, this will be done by loading the amplified DNA on a gel electrophoresis.
When the fragments are deemed okay they are sent to another research lab, in this lab the amplified sequences will be analysed on a fragment analyser. This fragment analyser uses capillary electrophoresis (CE), each fragment present in the capillary will be exposed to a laser. The fluorescent markers present on each fragment will absorb the light and emit it on a different wavelength, this emitted light will be recorded by the fragment analyser as a signal. These signals will be presented by peaks. The peak values are analysed using R studios, through R studios the clonality and similarity are calculated for the tested samples.
Genetic diversity is present inside the population and between the multiple populations, but there is not enough data to conclude a definitive conclusion about the genetic diversity of D. dichotoma.
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Katleen Van der Gucht
Ineke van Gremberghe