Informe de pràctiques de genòmica (GNM) (2017)Trabajo Inglés
Inclou l'informe de pràctiques de genòmica amb les il·lustracions pertinents corresponents a l'assignatura de Genòmica.
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Natalia Mingorance García
3r Biologia – UdG
Course 2016 – 2017
Professors of Genomics
Alba Abras and Melania Agulló
Faculty of Sciences
University of Girona
Natalia Mingorance García
3rd of Biology
Girona, March 19th 2017
Word count (excluding front page and bibliography): 2,006
Natalia Mingorance García
3r Biologia – UdG
Rh blood factor
The Rhesus factor is the most important protein-based blood group system and although Rh
is codified by more than one gene, the major Rh antigens are located on two Rhesus
proteins (RhD and RhCE) and are produced by differences in their protein sequences1.
According to this, the phenotypes for this gene are Rh D positive and Rh D negative and it is known that the Rh D gene is completely absent in Rh D negative individuals2.
Therefore, the genetic architecture of the Rh blood factor phenotypes has been determined as an intron size event. On top of that, it is known that most eukaryotic genes have their coding sequences (called exons) interrupted by noncoding sequences (called introns).3 Material and Methods The experiment was divided in two main parts: first of all, to extract the DNA from fresh blood samples (from H. sapiens) it was used a phenol-chloroform extraction protocol.
It’s important to consider that some of the components used on the DNA extraction were inhibitors of the PCR hence and were carefully removed after achieving their goal. They were hemoglobin, phenol and ethanol. In the first case, the TE buffer (which is a hypertonic solution) was used to prevent blood clotting and made lysis red blood cells, which contain hemoglobin and is precisely one of many inhibitors of PCR. Then phenol (which is toxic) was used to eliminate proteins and DNA and salts from the samples were precipitated using ethanol.
Another important step of PCR method was the hybridization temperature, which was specific of each species because if was higher than the optimal it could not amplify anything and if was lower it would amplify useless fragments. In that case, the optimal temperature was 65 ºC.
The second part consisted on a PCR and a posterior gel electrophoresis to analyze the samples (dividing DNA fragments of varying sizes). PCR reactions specifically used a forward primer within exon 4 and a reverse primer in exon 5 to amplify across intron 4 (see Figure 1). The primers were common to both D and CcEe genes2 and that matched with the genetic event of introns length. On the other hand, the electrophoresis method included a negative control (which was miliQ water) to check that all the previous steps were not contaminated.
2 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS Figure 1. PCR amplification across intron 4 of the Rh D and CcEe genes. 2 Results and Discussion The expected results were, on the one hand, a PCR product of 1,200 bp (which serves as an internal control and proofs the proper functioning of PCR) should have been amplified from Rh D positive samples as well as from Rh D negative samples. On the other hand, a PCR product of 600 bp (which is the diagnosis band) should have been amplified from Rh D positive. So the PCR product from the D gene was 600 bp shorter than the CcEe gene due to a deletion of the D gene (which was used in intron 4 to distinguish the D from CcEe genes)1.
The observed results were very disappointing because any PCR product was amplified. One of the possible reasons of these results may be that probably anyone had extracted enough blood to have the sufficient amount of DNA to analyze or to amplify by PCR method. Another possible cause could be that some component had inhibited the PCR. The negative controls were clean so it can’t assure that PCR was correctly working because negative controls are used for check if there’s any contamination (and it didn’t amplify anything).
A possible way to improve the PCR technique in this case would be trying doing the same process but with a previous purification of the primers, or using new primers –because maybe the ones used on this experiment were degraded during storage4–.
Another option could be using a kit of cleaning and purifying DNA which removes the phenol, proteins and cations residues. If the cause of the lacking PCR bands was not the absence of DNA, a dilution of the samples could be done so the inhibitors of PCR were diluted as well (on the condition that there was enough DNA for doing so)5.
3 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS Chondrodysplasia in dogs Introduction Processed pseudogenes are copies of messenger RNAs that have been reverse transcribed backwards into DNA and inserted into the genome. They generally lack introns, end in a 3’ poly A, and are flanked by target site duplications6. The fgf4 retrogene is a processed pseudogene that is located in chromosome 18 of dogs7.
In the case of fgf4 retrogene, the result of its belonging is expressed as chondrodysplasia7, which is an illness that makes dogs suffer from disproportionate short stature dwarfism of varying severity8.
So, the genetic event that has been determined here is processed pseudogenes that produce an expressed retrogene (fgf4) which phenotypes are both chondrodysplastic (short legs) and non-chondrodysplastic (long legs).
It is also known that a 5 Kb insert is found only in chondodysplastic breeds and it contains the conserved fgf4 retrogene7. The 3’ UTR and poly-A tail characteristic of retrotransposition of processed messenger RNAs was present in the insertion7 and, as previously exposed, it constitutes another proof that this is a processed pseudogenes genome event.
Material and Methods The experiment was divided in two main parts: first of all, to extract the DNA from fresh blood samples (from two different species of dogs) it was used a phenol-chloroform extraction protocol.
As discussed in the Rh blood factor instance, the PCR is a very sensible technique and it has lots of inhibitors. All of them were taken in account along the process.
It was also commented before that the temperature of hybridization of PCR samples was specific of each species. In that case was 47 ºC.
The second part consisted on a PCR and a posterior gel electrophoresis to analyze the samples (separate the DNA fragments of varying sizes). Concretely, the PCR reactions used a forward and a reverse primer to amplify a region of chromosome 18 of fgf4 retrogene (see Figure 2). On the other hand, the electrophoresis method included a negative control (which was miliQ water) to check that all the previous steps were not contaminated.
4 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS Figure 2. PCR amplification of a region on chromosome 18 of the fgf4 retrogene.7 Results and Discussion On the one hand, the expected results were a PCR amplification band of 132 bp in nonchondrodysplastic dogs and a band of approximately 5,000 bp in chondrodysplastic dogs.
Therefore, the dog 1 was supposed to be chondrodysplastic (meaning that it was supposed to show a 5,000 bp band) and the dog 2 was supposed to be non-chondrodysplastic (meaning that it was supposed to show a 132 bp band).
The observed results were that the biggest band didn’t appear. This was due to the fact that PCR couldn’t amplify this specific fragment of DNA because of its length, inasmuch as the Taq polymerase couldn’t amplify it. For that reason, all the results concluded that both of the two different species pertained to non-chondrodysplastic dogs. Also the negative controls were clean (there wasn’t any contamination of the samples).
In order to improve the PCR technique one option is to use specific primers that amplify a shorter region (or the entire insert) and obtain a band in the gel electrophoresis7. Other less preferable options may be letting the samples more time in the gel electrophoresis to get the band below and well separated or what’s more, increasing the agarose concentration of the gel to difficult the progress of the DNA band through the gel, such as raising the time of the electrophoresis but obtaining in the same size of the gel more separated bands.
5 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS Color in sheep Introduction Although there are over 300 genes identified with known roles in mammalian pigmentation9, the agouti signaling protein gene (ASIP) has been identified as a major regulator of pigmentation in many animals10. In this study, the focus was placed on sheep coat-color patterns which involve multiple alleles of ASIP gene. The phenotypes for this gene are white and black sheep.
In particular, ASIP gene encodes a signaling protein (ASIP) that can act as an antagonist of the melanocyte-stimulating hormone (a-MSH), producing a cascade of reactions that inhibits the formation of eumelanin and results in lighter coat color9.
Different studies have shown that recessive mutations in ASIP result in more darkly pigmented phenotypes whereas the white color is caused by deregulated expression of the agouti protein and results from a 190 kbp genomic duplication9. Also ASIP expression was not detected in the skin tissue of recessive black sheep with single copy functional ASIP alleles9.
Therefore, the genetic architecture of the color in sheep has been determined as a nonprocessed pseudogenes genetic event, because a non-processed pseudogene is a copy of a functional gene that may arise as a result of a gene duplication event and subsequently acquire mutations that make it become non-functional11.
Material and Methods The experiment was divided in two main parts: first of all, to extract the DNA from fresh blood samples (from different species of sheep) it was used a phenol-chloroform extraction protocol.
As mentioned in the previous cases, the PCR is a very sensible technique and it has lots of inhibitors. All of them were taken in account along the process.
It was also commented before that the temperature of hybridization of PCR samples was specific of each species. In that case was 60 ºC.
The second part consisted on two different PCR and a posterior gel electrophoresis to analyze the samples (separate the DNA fragments of varying sizes). Concretely, one of the PCR reactions used a forward primer called Agt16 and a reverse primer called Agt17 to amplify one region of the ASIP gene which can cover the 5’ breakpoint sequence (see Figure 6 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS 3)9. Otherwise, the Agt16 forward primer and the Agt18 reverse one were used in the other PCR reaction to amplify one region of the ASIP gene which includes the junction between the duplicated copies (see Figure 3)9.
Furthermore, the electrophoresis method included a negative control (which was miliQ water) to check that all the previous steps were not contaminated.
Figure 3. Structure of the ASIP gene duplication in genomic DNA.9 Results and Discussion Primarily, the expected results were a PCR amplification band of 238 bp in both white and black sheep for the 16-17 primers, one band of 242 bp in white sheep for the 16-18 primers and a lack of band in black sheep for the 16-18 primers. Therefore, sheep 2 was presumed to be white (meaning that it would show both bands in the correspondent place) and sheep 3 to be black (so it would only present the 238 bp band for the 16-17 primers). Furthermore, the sheep 4 and 5 were mouflons and supposed to be black.
In addition to this, here the 238 bp band acts as a control band and it’s always expressed in both phenotypes for the primers 16-17. However, the 242 bp band acts as a diagnose band, meaning that if the phenotype is black this band will appear and if it’s a white sheep it won’t.
The observed results were that the two bands appeared in sheep 2 and sheep 3 and the interpretation was that they were white (some of the samples had the negative controls contaminated and these results were directly refused). On top of this, it’s actually known that sheep 3 is heterozygote for another gene that confers coat pigmentation. Also, they were supposed to be black –while the data showed that this sheep was white–. Moreover, the observed results were that the two bands appeared in sheep 4 and sheep 5 and the negative controls were contaminated, so these results were rejected because they were a sign of contamination (in some step an error was committed) thus being an incongruous conclusion.
To improve the PCR technique, in this case, one option would be trying other specific primers for the gene that is causing the coat pigmentation of this sheep species (with a previous search of information).
7 Natalia Mingorance García 3r Biologia – UdG UNYBOOK: nattymg23 GENOMICS Bibliography 1: Flegel, W. A. 2007. The genetics of the Rhesus blood group system. Blood Transfusion 5(2), 50–57. doi:10.2450/2007.0011-07.
2: Hyland, C A, L C Wolter, and A Saul. 1995. Identification and Analysis of Rh Genes: Application of PCR and RFLP Typing Tests1. Transfusion medicine reviews IX: 289-301.
3: Alberts, B., Johnson, A., Lewis, J., et al. (2002) Molecular Biology of the Cell (4th ed.).
New York: Garland Science.
4: Fekete, A., Bantle, J.A. (1991). A simple, efficient method for purification of degraded PCR primers. Biotechnol Tech 5 (6): 479-482. doi:10.1007/BF00155498.
5: Ramakers, C., Ruijter, JM., Deprez, RH., Moorman, AF. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 339 (1): 6266. doi:10.1016/S0304-3940(02)01423-4.
6: Kazazian, H. H. (2014). Processed pseudogene insertions in somatic cells. Mobile DNA 5, 20. doi:10.1186/1759-8753-5-20.
7: Parker, H G, B M VonHoldt, P Quignon, E H Margulies, S Shao, D S Mosher, T C Spady, et al. 2009. An Expressed Fgf4 Retrogene Is Associated with Breed-Defining Chondrodysplasia in Domestic Dogs. Science 325: 995–98.
8: Kyöstilä, K., Lappalainen, A. K., & Lohi, H. (2013). Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10. PLoS ONE 8 (9), e75621. doi:10.1371/journal.pone.0075621.
9: Han, J L, M Yang, Y J Yue, T T Guo, J B Liu, C E Niu, and B H Yang. 2015. Analysis of Agouti Signaling Protein (ASIP) Gene Polymorphisms and Association with Coat Color in Tibetan Sheep (Ovis aries). Genetics and Molecular Research 14: 1200–1209.
10: Norris, B J, and V A Whan. 2008. A Gene Duplication Affecting Expression of the Ovine ASIP Gene Is Responsible for White and Black Sheep.” Genome Research 18 (8): 1282– 93. doi:10.1101/gr.072090.107.
11: Tutar, Y. (2012). Pseudogenes. Comparative and Functional Genomics, 2012 (2012): 14. doi:10.1155/2012/424526 8 ...