Simple and Cost-Effective Method for Sex Determination - Lab Report Example

A simple and cost-effective method for Sex determination, with PCR, using ovine amelogenin (intron 5) - specific Name Institution Date A simple and cost-effective method for Sex determination, with PCR, using ovine amelogenin (intron 5) - specific Introduction Due to its sex specific sequence variability, the amelogenin gene has been utilised in determining sex within the family of Bovidae (Dervishi et al, 2008, p.241). The amelogenin gene encodes protein involved in the production of teeth enamel (Bansal, 2012, p. 395). This gene is highly conserved in vertebratas and the amelogenin gene is found within X-chromosome and Y-chromosome and there is highly homology between the two alleles (93% in cows). Amelogenin contains approximately 90% of the total enamel matrix proteins and significantly contributes to the mineralization and morphological changes within enamel. The location of the human amelogenin gene is on X-chromosome at Xp22.1–p22.3 and on Y chromosome at Yp11.2 (Bansal, 2012, p. 395). 90% of the transcripts expression occurs on X-chromosome while 10% occurs on Y-chromosome. The X and Y replicas of amelogenin gene do not go through homologous recombination and hence this makes amelogenin gene the most preferred genetic market when determining sex (Bansal, 2012, p. 395). Accordingly, this report aims at investigating sexing unknown samples through PCR, using ovine amelogenin (intron 5) - specific primers. Objectives The main objective of this experiment is to simultaneously amplify specific primers corresponding to Y- and X-amelogenin gene in order to determine a reliable, reproducible and efficient PCR-based sexing of unknown samples (Chen, Xu & Yu, 2007, p.1689). Methodology PCR ingredients All chemical s were provided by the University. PCR ingredients included: A labeled tube; 5X BUFF 5X DNA polymerase buffer including magnesium ions; dNTP;10 mM mixture of 4 dNTPs; Sterile water; Forward ovine amelogenin gene primers (intron 5 specific); Reverse ovine amelogenin gene primers (intron 5 specific); and A, B, C & D Template ovine genomic DNA (unknown gender) (2). PCR set up All unknown sample templates were labeled using initials and then the above reagents were added to both unknown tubes within the amounts shown below: Unknown sample tubes contain 7.0 ul; Sterile water (H2O) 6.4 ul; 5X DNA Pol. Buffer (5X BUFF) 4.0 ul; 10 mM dNTP mix (dNTP) 0.4 ul; AML Forward Primer (F) 1.0 ul; and AML Reverse Primer (R) 1.0 ul. Lids of the tubes were then closed and labeled with my initials on all lids. After this, tubes on ice were taken to the instructor (PM) and the following reagents were added; Phusion DNA polymerase 0.2 ul and total reaction volume 20.0 ul. Making the gel In making the gel, approximately 35 ml of the gel was used. Buffer was used to ensure adequate volume 50 of Tris Acetate EDTA (TAE). For a 1.5% gel 750mg was dissolved within the 50 ml of TAE buffer through boiling it. The molten gel was allowed to cool below 50 0C and a small amount of (˷1ul of 10mg/ml) of ethidium bromide was added and mixed in. The gel was then poured, while still in liquid form, into a suitable gel mould and well-forming ‘comb’ placed at the top. The gel was then allowed to cool and set. After setting the gel into the mould, it was then submerged in TAE buffer within an electrophoresis tank Sample preparation After the completion of the PCR, 4.0ul of 6x bromophenol blue dye buffer was added to every 20 ul sample. 20 ul of every sample was loaded onto the gel after it was removed from the well-forming ‘comb’ Electrophoresis During electrophoresis, the lid was placed on the tank. Electrodes were then connected to the power pack and power was maintained below 100 volts/100mAmps. The electrophoresis continued for about 30 minutes until the dye-front moved about 2-4cm Visualization The DNA (PCR products) were visualized under ultra-violet light Results Sex determination of unknown samples For all unknown samples, female and male samples were tested and analysis of PCR products was done using gel electrophoresis and expected banding pattern (200 bp: male and 150 bp: female) was observed. Figure 1: Electrophoretic pattern of amplified fragments for group 1 Figure 2: Electrophoretic pattern of amplified fragments Figure 3: Detail illustrating amplicons of amelogenin gene fragment It was observed that there was a strong band from the X-linked marker but the Y-chromosome marker had a weak band. Sensitivity test showed that for template DNA with low molecular weight ladder, all the sexing markers amplified all the replicate PCRs. As projected, from the ovine sequence, a 200 bp and 150 bp product, and this represented amplification from both the X and Y chromosome amelogenin was detected for the sample. In addition, results indicated that female samples revealed one product while male samples revealed two bands. This occurred in results from both groups. The following ladder was used to size and approximate quantification of the DNA. The ladder enabled purification and high quality of DNA bands and hence no double peaks were observed during electrophoresis and on the gel. This also allowed precise calculation of the precise amount of DNA. Figure 4: SM1191 Low molecular wt Ladder Discussion The aim of this experiment was to investigate the protocol for sexing unknown samples in order to determine their sex. Basing on the amelogenine gene located on X and Y chromosomes, specific primers was used and PCR was established in amplification of 150 bp fragment from Y chromosome for the male and 200 bp fragment from X chromosome, for the female. In this study, results from both groups demonstrated a high DNA fragmentation pattern (150-200 bp) and this demonstrated good DNA preservation and hence the primer sets were efficient for determining sex in the unknown samples (Brenda et al, 2014, p. 2). According to Dervishi et al (2008, p. 245) currently there are molecular techniques used in determining sex. Amelogenin gene has been used in gender assignation because it can encode a vital protein within the developing enamel matrix of a mammal (Salabi et al, 2014, p. 2). Amelogenin gene has been used in determining sex in Bovidae family because this procedure does not require additional amplicons or restriction endonuclease process (Dervishi et al 2008, p. 245). Expected sizes of PCR products In this experiment, sex was determined using amelogenin gene. The sizes of the PCR products were between 150 bp and 200 bp. The expected size was 215 bp fragment specific for chromosome Y and 278 bp fragment specific for chromosome X. this is according to Grzybowski et al (2006, p. 112) who conducted a study on sex determination of cattle using amelogenin. The testing for homogametic sex is supposed to be always homozygous while heterozygous phenotype is supposed to show for heterogametic sex (Grzybowski et al, 2006, p. 112). This was evident in this experiment where the heterozygous phenotype appeared for heterogametic sex samples. In addition, the PCR products specific for chromosomes Y and X should have constant position within a separating medium that matches the fragment size and this was evident in the experiment where there was correspondence between fragment size of 150 bp and 200 bp (Trigal et al, 2012, p.355). Amplification of the gene sequence was adequate in establishing the sex in this experiment just like it has been successfully performed in ovine embryos sexing studies (Ballin & Madsen, 2007, p. 386). Blank results There were some blank results and this could have resulted from contamination of the DNA or the ladder. Contamination can occur due to failure to use sterile water and pure reagents or not sterilizing tubes. In addition, blank results could have resulted from inaccurate estimate of concentration from the gel or inaccurate quantitation of the DNA (Terri & Bessetti, 2005). Distinguishing between male and female Male and female can be distinguished by the number of bands that form after PCR (Gokulakrishnan et al, 2013, p.33). A study conducted by Gokulakrishnan et al (2013, p.33) showed that PCR products of meat samples after electrophoresis indicated 2 bands for male tissues and only 1 band for female sample. Therefore, in this experiment determining the sex of the samples was a bit complex. The location of amelogenin gene in mammals is on X-chromosome and Y-chromosome. This is in line with Pajares et al (2006, 445) whose study showed that all female samples had 1 band while male samples indicated 2 bands. However, the length of the two fragments can be used in distinguishing the gender. The distinguishing feature of length polymorphism between X-chromosome and Y-chromosome has been utilized as the target to determine the gender in mammals (Mace & Roy, 2008, p. 3). Therefore, the fragment with 200 bp was identified as the female while the fragment with 150 bp was identified as the male since studies have shown that females have longer fragments when compared to male fragments Pajares et al (2006, 445). Other methods used for sexing Other methods that have been used in determining gender include PCR method utilizing sexual dimorphism between the Zinc Finger-X and -Y (ZFX-ZFY) gene as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for mitochondrial DNA (mtDNA) cytochrome B (CYTB) (Pfeiffer & Brenig, 2005, p.3). Just like in PCR using amelogenin gene, results of these methods indicated noticeable band patterns between female and male DNA where Male DNA sample produced twp PCR bands while female DNA produced one band (Kim et al, 2016, p.61). Comparing sheep results with Chen et al in cattle According to Chen et al (1999, p. 211), the protocol for sexing bovine using PCR amplification of bovine amelogenin gene on the Y and X chromosome on cattle was successful. According to the study the DNA sequencing demonstrated 45.1% homology. According to the study results, there was a single 467 bp fragment and this was deduced as the female while there were 2 fragments on the 467- and 341-bp products and this was deduced to be the male (Chen 1999, p. 214). However, in this experiment there was one fragment for the male and one fragment for the female. Therefore, the results of this experiment did not have correlation with the results of Chen et al (1999), in regard to the number of the product’ fragments. Nonetheless, just like in Chen et al (199), the size of the female (X-chromosome) was longer than the size of the male (Y-chromosome) which indicates correlation between the results of this experiment and Chen et al (1999). Use of complex thermocycler program in the Chen et al The complex thermocycler was used in amplification of DNA through polymerase chain reaction (PCR). The complex machine was used to facilitate temperature sensitive reactions such as restriction enzyme digestion. This is because the machine has a thermal block that has hole where tubes that hold reaction mixtures are inserted. Consequently, the thermocycler increases and decreases the temperature systematically. Other uses of the technique The technique is also used in human identity testing applications than include forensic DNA analysis. Conclusion It has been determined that the method used in the experiment is simple and accurate in determining the sex of unknown samples based of PCR procedure, using bovine amelogenin primer sequences. The findings in this experiment indicate that PCR assay using ovine amelogenin is reliable in sex identification in sheep and other animals as well. Because of comparatively short size (less than 200 bp) of the PCR products, the method can be used in determining sex of animal samples that have highly degraded DNA within comparatively shorter time duration. In addition, this method does not involve any control amplicons with second locus-specific autosomal primer pair. Moreover, restriction endonuclease steps are not necessary when determining sex and controlling the PCR reaction. This experiment demonstrates that the amelogenin gene can be utilized in determining sex, with a high efficiency and accurateness. Reference list Ballin N & Madsen K, 2007, Sex determination in beef by melting curve analysis of PCR amplicons from the amelogenin locus, Meat Science 77(1), pp: 384–388. https://www.researchgate.net/publication/51781743_Sex_determination_in_beef_by_melting_curve_analysis_of_PCR_amplicons_from_the_amelogenin_locus Bansal A, Shetty D, Bindal R & Pathak A, 2012, Amelogenin: A novel protein with diverse applications in genetic and molecular profiling, J Oral Maxillofac Pathol, 16(3), pp: 395–399. < https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519216/> Brenda A, Linda M & Montiel R, 2014, Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis, PLoS One, 9(8): e104629. < https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123986/> Chen A, Xu Z & Yu S, 2007, Sexing Goat Embryos by PCR Amplification of X- and Y- chromosome Specific Sequence of the Amelogenin Gene, Asian-Aust. J. Anim. Sci, 20(11), pp : 1689 – 1693. < http://ajas.info/upload/pdf/20-234.pdf> Chen C, Hu L, Wang H, Wu K, Hung M & Cheng W, 1999, Gender Determination in Single Bovine Blastomeres by Polymerase Chain Reaction Amplification of Sex-Specific Polymorphic Fragments in the Amelogenin Gene, Molecular Reproduction and Development, 54(1), pp:209–214. Dervishi A, Martinez-Royo A, Sanchez P, Alabart J, Concero M, Folcj J & Calvo J, 2008, Reliability of sex determination in ovine embryos using amelogenin gene (AMEL), Theriogenology, 70 (2008), pp: 241–247. Grzybowsk G, Prusak B & Romaniuk B, 2006, A novel variant of the amelogenin gene (AMEL-X) in cattle and its implications for sex determination, Animal Science Papers and Reports,24(2), pp: 111-118. Gokulakrishnana P, Kumara B, Sharmaa S, Mendirattaa P, Malava D, Sharma D, 2013, Determination of sex origin of meat from cattle, sheep and goat using PCR based assay, Small Ruminant Research journal, 113(1), pp:31-33. Kim Y, Kang S, Jeong D, Cho I & Han S, 2016, Molecular Sexing and Species Identification of the Processed Meat and Sausages of Horse, Cattle and Pig, J. Emb. Trans, 31(1), pp:61-64. https://www.researchgate.net/publication/305803488_Molecular_Sexing_and_Species_Identification_of_the_Processed_Meat_and_Sausages_of_Horse_Cattle_and_Pig. Mace M & Roy B, 2008, A highly polymorphic insertion in the Y-chromosome amelogenin gene can be used for evolutionary biology, population genetics and sexing in Cetacea and Artiodactyla, BMC Genetics, 9(64), pp:1-9. Pajares G, Álvarez I, Fernández I, Pérez-Pardal P, Goyache F& Royo L, 2007, A sexing protocol for wild ruminants based on PCR amplification of amelogenin genes AMELX and AMELY (short communication) , Arch. Tierz., Dummerstorf, 50(5), pp: 442-446 Pfeiffer I & Brenig B, 2005, X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European red deer (Cervus elaphus), BMC Genet, 6: 16. < https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1079817/> Salabi F, Nazari M & Cao W, 2014, Cell culture, sex determination and single cell cloning of ovine transgenic satellite cells in vitro, Journal of Biological Research-Thessaloniki, 21(22), pp: 1-11. < http://download.springer.com/static/pdf/537/art%253A10.1186%252Fs40709-014-0022-z.pdf?originUrl=http%3A%2F%2Fjbiolres.biomedcentral.com%2Farticle%2F10.1186%2Fs40709-014-0022 z&token2=exp=1490845696~acl=%2Fstatic%2Fpdf%2F537%2Fart%25253A10.1186%25252Fs40709-014-0022 z.pdf*~hmac=1134a5754af8bd820fe3121d418058088e3a29d0ce4e800f3b771ffebc513f35> Terri S & Bessetti J, 2005, Identifying and Preventing DNA Contamination in a DNA-Typing Laboratory. https://www.promega.com/-/media/files/resources/profiles-in-dna/802/identifying-and-preventing-dna-contamination-in-a-dna-typing-laboratory.pdf?la=en Trigal B, Diezi G, Monozi C et al, 2012, Comparative study of PCR-sexing procedures using bovine embryos fertilized with sex-sorted spermatozoa, Spanish Journal of Agricultural Research, 10(2), pp: 353-359. < http://ria.asturias.es/RIA/bitstream/123456789/3681/1/Comparative%20study%20of%20PCR-sexing.pdf> Read More