Determination of sex is the nurturing mechanism by which the sex of an embryo has been identified by the formation of gonad. (Nef and Vassalli., 2009). A pair of chromosome is present in all individuals but it is XX for female and XY for male. By this chromosomal pair, male phenotype acts as a heterogametic as it contains XY and female acts as a homogametic due to the presence of XX chromosome. (Daneau et al., 1995).Gene action, interaction within the embryo and the maternal environmental interactions all decides the formation of the male and female reproductive systems and these interactions induces the difference between the chromosomal sex and phenotypic sex.( Michael and cummings, ) Testis determining factor is a gene present in Y chromosome that involved in the developing and control mechanism , that accelerate specific gene, that induces signal for the male gamete formation. Absence of TDF leads to the formation of ovaries as the signal is not received. Thus, TDF acts as a growth switch for sex examination. (Daneau et al., 1995). Primer is the specific and short fragments of DNA molecules and acted as the template initiated for the replication of new stand. (Newton and Graham, 1994).
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Y chromosome contains nearly 40 to 50 genes. Stimulation of some specific gene on male chromosome is used to determine the sex in mammals and birds. In Y chromosome, presence of SRY gene plays an important role in determining the sex. This SRY gene activation leads to the male testis development. SRY gene encodes a protein of high mobility group family. This HMG comprises of DNA binding protein and nuclear localizing signal and that protein plays a main role in formation of testis from the primitive gonadal tissue after receiving a testis determining signal via TDF. Absence of TDF results in the formation of ovaries from the primitive gonads. Zinc-finger transcription factor is encoded by the SRY gene. (Hossain and Saunders., 2001) Polymerase Chain Reaction (PCR) is an invitro technique which is used to amplify the specific DNA sequences. In Biotechnology field, the application of PCR is widely used in Molecular biology, (DNA sequencing, probe generation), clinical use, detection of mutagenesis and forensic science.(Becker et al., 1990). PCR is the invitro DNA technique which produces the large number of specific DNA between regions of the two known DNA sequence. DNA polymerase enzyme plays a key role to produce more copies of own DNA template and this mechanism sustained for many cycles and leads the DNA to exponentially amplified..(Newton and Graham,1994 ).DNA is the structural function of all livings organisms which carry genetic material from one to next generation.DNA is used widely in the medical and research areas.(Rosalind Franklin University of medicine and science 2004).Identification, isolation and quantification of DNA decides the DNA purity.(Promega.,2009).Concentration of DNA purity is enhanced by extracting DNA from the cell, separating it from RNA and protein and it is followed by quantification and analysis using spectrophotometer. DNA absorbs the ultraviolet rays at 260nm and it leads to the understanding of concentration and purity of DNA. (Becker et al., 1990).
LOCATION OF SRY GENE IN Y CHROMOSOME:
Agarose gel electrophoresis technique is widely used to separate the biological molecules based on its size, charge and shape. Ethidium bromide acts as an intercalating agent to view the bands under UV rays by binding it into the specific molecules.(Becker et al., 1990). As the DNA is negatively charged molecule, it passes towards the positive charge in the agarose gel under the influence of specific voltage. Small molecules migrate fastly than the molecules with high molecular weight.
To identify the sex of given three unknown sample using the polymerase chain reaction performance.
MATERIALS AND METHODS:
Buffer(AL, AW1,AW2, AE and TAE),Ethanol, Vertex Machine, Incubator, Centrifuge Machine, QIAmp Mini spin column, Micropipette, Micropipette tip, Cuvette, Primers(Forward and Reverse of SRY and GAPDH), Thermocycler, 3 unknown sample, positive control, Negative control, Ethidium bromide, Agarose solution, Powerpack , Electrophoresis tank, UV light imager, Quartz glass cuvett, unknown sample 4a, 4b and 4c, dNTPs, Taq polymerase enzyme and Mg2+ ion.
To purify the DNA from the specific tissue, we have added 200Âµl AL buffer to the given sample and the proper mixing was handled by vortexing for 15 seconds and then incubate the mixture for 10 minutes at 70 degree C. Then we added 200 ul of ethanol to this sample and mixed it well using vortexing for 15 S to enhance proper precipitation and transfer the obtained lysate into new QIAmp Mini spin column and it is followed by the process of centrifugation at 13000 RPM for 1 minute to allow the column to pass through. Transfer the availed column into the new 2ml collection tube and discard the tube with filtrate. To the collected column, we have added 500 ul of Buffer AW1 and it is followed by centrifugation at 8000 RPM for 1 minute. Then we transferred the acquired column to the new 2ml collection tube and added 500 ul of AW2 to that and allow it to centrifuge at 3000 RPM for 3 minutes. Discard the filtrate and centrifuge the column again at 3000 RPM for 3 minutes .Again discard the collected filtrate and centrifuge the obtained column at 13000 RMP for 1 minute and then transferred the column into new 1.5 Ml collection tube. Added 200 ul of buffer AE to this column and incubated it in room temperature for 1 minute and it is followed by the centrifugation at 8000 RPM for the duration of 1 minute. Gone over this step once and removed off the column and kept the 1.5 ml tube on ice to proceed with the PCR set up procedures.
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POLYMERASE CHAIN REACTION:
We have taken 6 thin walled, fragile microcentrifuge tubes with 0.2 ml capacity and label it as '-' , 'P', 'N','1', '2' and '3' respectively. Prepared a master mix using buffer, primers, water, dNTPs, enzyme Taq DNA polymerase, Mg2+ ion. Given primer contains both forward and reverse SRY and GAPDH genes. Equally divided these master mixtures into 6 and poured into all the 6 tubes and this has to be done on the behalf of the control and unknown sample.
We have made the master mixture and poured it on the tube '-' using the proposition below.
Master Mix Volume(Âµl)
Final Tube volume(Âµl)
Added 8 ul of DNA samples to all the given 5 tubes to achieve a 20 ul in whole. Pour 12 ul of the prepared master mix in each 5 tubes followed by the addition of 8 ul of sample DNA.Then these tubes were kept in the thermocycler and position of each group has been noted. The PCR thermal cycle involves several steps as follows: The main three steps involved are: Denaturation, annealing and extention.
To separate the double stranded DNA into single stranded, (i.e) to unwind the double stranded DNA, we were dealing with the process of initial denaturation . To proceed with this denaturation process, set the thermocycler in 95 degree C up to 5 minutes. Carried out with the following procedure for 40 cycles. Process of denaturation were done at 94 degree C for the duration of 30 s. Annealing step consumes 30 s at 60 degree C and extension requires 72 degree C up to 1 minutes. At last, final extension procedure is carried out and this has been done at 72 degree C for 10 minutes. After recycling, retain it at 4 degree C and arranged the temperature at 105 degree C to avoid evaporation of the content present in the tube. Then store the samples at 20 degree C for future usage.
AGAROSE GEL ELECTROPHORESIS:
To separate the fragments of DNA and to view it, we were dealing with the electrophoresis process. We poured 150 ml of agarose gel into the conical flask and it is followed by the addition of 10ml of ethidium bromide solution. We assigned the mini-gel electrophoresis tank and in middle, we placed the 8 sample comb. Dispense the prepared gel up to 5mm depth and allowed it to set for 15 minutes. Allowed the gel to set and simultaneously, we added 20 ul of sample loading buffer to all 0.2ml tube which contains 20ul of DNA. Removed the comb after proper gel setting and the TAE electrophoresis buffer was poured over that with 0.5cm breadth. Carefully, Loaded 20ul of each samples (Tubes "-", "P", ''N'',''1'') to specific distinct wells and the addition of 4ul DNA ladder were loaded on separate well. We have loaded the samples in M, P, N, 1, 2, 3 position. Sample positions over the lanes were noted. Electrical connection has to be given to this prepared electrophoresis system with sample. It paves the way for the gel to run at 100V for 40 minutes or else allow it to run until the migration of the blue gel towards the half way down of the gel occurs.
Running of the dye takes place up to 600bp respectively. Then place the gel under the UV light and the images were clearly viewed with the help of ethidium bromide stain. Note down the appeared image of the gel. (Spikings et al., 2010)
Determination of DNA can be accomplished by specific feature and characterization to DNA, specifically at 260 nm absorbance while for protein, it is 280nm absorbance.
We Poured 1 ml of distilled water into the Quartz glass cuvette and placed into the spectrophotometer. Allowed the spectrophotometer to switch on and accelerate the run button. Absorbance of the spectrophotometer was imaged already at 260 nm and 280 nm. To the 1ml of distilled water, 20 ul of 4a DNA solution were added. And switch on the run button. Readings are displayed over the spectrophotometer for 4a DNA sample at 260 nm and 280 nm absorbance. Likewise readings for 4b DNA and 4c DNA can be taken out. Record the displayed reading for all the DNA samples. (Spikings et al., 2010).
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Sex determination by SRY gene using polymerase chain reaction has shown in the figure below.
B U1 U2 U3 M F SM
Figure illustrates that, DNA bands has been visualized by absorbing ethidium bromide in the given solution. The pair of primers (SRY and GADPH- Housekeeping) were utilized for the amplification of 3 given DNA samples. Lane B stands for blank (no DNA), Lane U1 indicates unknown DNA 1 and lane U2 stands for unknown DNA 2 and lane 3 referred for unknown DNA 3 . M indicates male and it stands as a SRY Positive DNA, lane N for SRY negative DNA, and lane SM acts as a DNA size marker(ladder). Arrow at the margin shows the direction of DNA migration. DNA marker present in the gel is not clear and it is looking staggered because of the formation of smear. It contains several bands between 300 and 1200bp. But the bands visualizing are seems to be very mild. Positive control contains only one band at 200bp and it seems to be very mild. Likewise, negative control elicits single band formation at 200bp. Lane unknown 1 has 4 bands at 200bp, 400bp, 600bp and in 800bp respectively. Lane 2 with unknown sample shows 2 band formation at 200bp and 400bp. Lane 3 with unknown sample 4c contains 4 bands between 200bp, and 800bp respectively.
We expected the band in a proper manner which is corresponding to the positive lane. But DNA marker was not clear and looking staggered due to smear formation. Positive control has two bands at 200bp and 600bp. It appeared very mild band and half of the region not cleared. Negative control had 2 bands and approximately presented at 200 and 300bp respectively. One band was looking bright and thick and another one band was thin and light. Lane 1 has 4 bands, two bands were bright and another two were mild thin. 1,2, and 3 bands were presented approximately at 100 to 300bp. Lane 2 had two bands presented at 200 and 300bp. Blank had two bands and approximately presented at 100 and 20bp. One band was bright and another was mild. In this result, we could not found the presence of Male gene as the lane with positive control is not cleared.
DNA DETERMINATION :
Determination of DNA was examined from the provided unknown samples 4a, 4b and 4c DNA using the spectrophotometer and the absorbance were read at 260 nm and 280 nm respectively. Calculated ratio has been shown in the below mentioned table.
Ration Abs260 /Abs280
DNA concentration in the 1 ml(Âµg DNA ml)
The Dilution factor for the sample
DNA concentration in the 20Âµl sample (Âµg DNA ml)
The amount of DNA in the 20 Âµl sample(Âµg)
Ratio of sample 4a absorbance at 260nm and 280nm was ----------.Absorbance of pure DNA ratio Abs260/280 was ____. So Sample DNA __was the pure DNA when compared to other 2 DNA samples.
Sex chromosome is present in all the organisms and it enhances the formation of sex organs in each and every organism. A gonad requires 2 X chromosomes for the development of ovary, whereas presence of some specific gene in Y chromosome leads to the development of testis.
This method depends mainly on Y chromosomal sequences. In this study, a basic and accurate procedure of sex determination has been carried out using a forward and reverse primer of SRY gene. SRY gene encodes for a main protein and that enhances the development of male phenotype. (Pfeiffer and Brenig., 2005) Chromosome Y carries the genetic information specific to the male sex. (Goodfellow and Badge., 1993).The elaboration of SRY gene sequences governing an important role in understanding the mechanism involved in sex determination. SRY (sex region of Y) gene is suspected to be the testes determining factor. Presence of SRY gene induces the determination of male sex by suppressing the Z gene. This leads to the formation of testis from indifferent gonads. (copelli et al., 2000 ) .The cloning of the bovine SRY gene is important as it acts as an alternative biological model system. (Daneao et al., 1995).The SRY gene expression occurs when testis initiated to form in the gonadal tissue and presence of germ cell is not required in this case. Zinc-finger transcription factor is encoded by the SRY gene. This regulates and control activity of other gene transcription by binding with the target sites of DNA molecule. The SRY gene provides instructions for making a transcription factor called the sex-determining region Y protein. GAPDH gene means glyceraldehydes-3-phosphate dehydrogenase, it sed as a marker for proliferation of cell. Control of Housekeeping gene expression is determined by this gene. (Wettasignhe et al., 2010 ) Main role of SRY gene is to promote testicle differentiation via specific gene activation and repressing the genes which is involved in ovary development. Next step of genes that functions by involved in the developing cascade leads to the understanding of some possible genes called as SOX9 gene. This gene encodes for a high-mobility group protein which promotes the differentiation of male and it expression in developing male gonad begins soon after the expression of SRY gene.(Genetics in medicine,2010)
Sex of an early embryo can be found only by using the revolutionized technique called Polymerase chain reaction. Sex identification in bovine embryos depends on the specific DNA amplification of Y chromosome by PCR. Polymerase chain reaction acts as a powerful tool and it is a sensitive method which influences the examination of male and female gamete (Lopatapova et al., 2008 ) Postnatal determination of sex has been enhanced due to the multiplication of specific genes in both X and Y chromosomes by the rapid polymerase chain reaction.( Majumdar.I.D et al., 2004 ) Gel electrophoresis was used to analyse the PCR product and also to detect the DNA bands size (Kobayashi and Hecker., 2000).
In our present experiment, among the 3 samples, two samples are purified and remaining one sample was unpurified. Purification of DNA sample involves the removal of unwanted cell molecules like RNA, protein, cell debris etc. With this pure DNA sample, we can enhance the Polymerase chain reaction.(Qiagen., 2010) According to our obtained gel image result , DNA bands absorbing the ethidium bromide which is present in the agarose gel acts as a intercalating agent and it paves the way for visualization of bands with the help of PCR. Bands we were visualizing in the gel are not clear as it faced some disturbance while pipetting and it leads to the contamination of the DNA sample. DNA marker has not much cleared because of improper pipetting at the time of sample poured into the ladder well. Positive control sample shows no proper band formation. It shows only one band in 200bp. Negative result for the given sample emphases several reasons. Ladder is seems to have more band formation but still it is staggered and the bands are not able to view in a genuine way, this might happen because of the gel solidification while loading it into the tank.
Positive control shows mild band formation at 200bp but it not formed in a correct manner and so it is difficult to examine its base pair sizes. This malformation has been occurred due to the contamination held by the presence of protein molecule and by RNA molecule. Unknown 1, 2 and 3 shows formation of bands but the positive and negative control elicits only one mild band formation at 200bp. As this positive control band is not stands corresponding to the provided unknown samples, we could not detect the sex of the given sample. Blank lane contains no DNA. Loading of gel in blank occurs only with sterile water. But in gel results, formation of band has been viewed at the specific blank site. It clearly displays that this is due to improper pipetting and contamination. (Spikings et al., 2010). Negative result might be happened due to the mistake occurs in concentration of gel, condition of the buffer, wrong measuring of the added sample contamination occurrence while pipetting, variation in voltage strongly affects the migration of DNA molecule in the gel.
DNA purity is determined at 260nm by using the spectrophotometer. For protein molecule, the absorbance is 280nm. Presence of RNA molecule and protein molecule most probably affects the purity of DNA sample. Some contamination and improper band formations all occurs due to the combination of protein and RNA in DNA molecule.
Purity of DNA is measured using spectrophotometer and it is calculated by Abs 260/280nm. (promega, 2009). Determination of DNA from the given samples 4A, 4B, 4C has been evaluated using spectrophotometer at 260nm and 280nm . Absorbance of sample 4a at 260 nm and 280 nm is_________.
The ratio of pure DNA is 1.8 in an absorbance of 260/abs280. Here we have got ---------for 4a DNA sample. So DNA 4a was the pure DNA among other samples provided. But it contains more impurities, so this sample is not suitable to carry out with PCR reaction.
Here, we have used pair of primers to amplify the specific target sequences from the provided samples. Provided pair of primers is bovine SRY and GAPDH and this includes both forward and reverse primers in the direction of 3' to 5' end and from 5' to 3' end. Each primer contains 24 sequences. SRY primer reaches 741 base pair in product size while GAPDH product size is 218 base pairs.
The sequences of the primers for Bovine SRY:
Product size (bp)
AAA CAG TGC AGT CGT ATG CTT CTG (24)
GCC TTT GTT AGC GAG AGT AAG GAA (24)
CCA ACG TGT CTG TTG TGG ATC TGA Â (24)
GAG CTT GAC AAA GTG GTC GTT GAG Â (24)
(Spikings et al., 2010).
In our present gel experiment, similar sequences of primers were appeared in the same base pair level, and this is due to primer dimer. This might be happen because of more addition of primers or else by self annealing process. Normally, blank has to show no band formation, but in our case we were visualizing mild band formation at the blank site. From this it is clear that the band formation in blank is due to irrespective formation of primer. Succesfull result from PCR highly depends on the quality of primer. Quality of good primer includes no binding with another primer. Quality of good primer has been evaluated using he primer software and this process elicits good results in polymerase chain reaction.(Mann et al,. 2009)
According to the obtained gel image results, bands of DNA has visualized by absorbing ethidium bromide present in the agarose gel using polymerase chain reaction technique.
The sex determination method is widely used to examine the Y chromosome and its role using polymerase chain reaction. This process greatly helps to identify the sex from the early embryo stage itself. This technique has good response in embryo transfer technology, and also it contains many advantages over biological and agricultural means. (Lopatorova et al ., 2008). Specific gene amplification from Y chromosome by polymerase chain reaction serves as a a powerful tool to determine the sex of calves in the early embryo stage itself. (Peippo et al., 2007).Female sex which carries XX acts as a default and that describes that the pattern of gene in females can be overviewed even in the absence of triggering signal (Piprek.R.P., 2009).
Sex determination have merit over embryo sexing, by that we can deal with the cytogenic analysis, function of DNA which is specific for Y and to understand about the male specific antigens. Determining the sex of developing embryo is used well in cattle breeding to alter the pregnancy. (Shan-Nan Lee., 2000). The current study reveals PCR dependent examination using markers specific for X and Y chromosome. (Ghosh and Dey., 2001).