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The sex determination in the biological system plays an important role in determining the sex of the individual at the embryo stage. Sex determination can be done in many ways. 1) Alfred Jost and his colleagues developed the surgical removal of the gonadal ridges from developing rabbit foetus. Since both the testes and ovaries are developed from the genital ridges, the removal of the ridges prior to the gonadal development results in female development. 2) The second method involves the genetic method of sex determination. The Y chromosome in males carries the information required to determine the male sex characteristics. The gene on the Y chromosome is called the TDY/Tdy (Testis Determining Factor). The Tdy gene is abscent for females. There are several other genes which are required for the formation of testis. On such gene located on Y chromosome is the SRY gene (Sex Determining Region Y gene). The SRY gene in humans is about 35Kb in length and it encodes for a protein called High Mobility Group (HMG). The High Mobility Group protein has DNA-binding properties and acts like a transcriptional regulator for the development of male characteristics (Goodfellow and Badge, 1993).
The reason behind determining the sex at the embryo stage has much clinical significance. Some of the X-linked inherited diseases may be transferred from the maternal blood to the foetus. The X-linked recessive traits are often expressed in males. The males require only a sing allele to be inherited to express the trait, whereas a female require inheritance of both the alleles. The earliest procedures for embryo sexing were carried out using various invasive techniques. But these techniques were too risky to the developing foetus. Pre-implantation genetic diagnosis for embryo sexing involves the risk of misdiagnosis (Goodfellow and Badge, 1993). And other method like Chorionic Villus Sampling (CVS) also had a foetal loss of 0.5-2% (Goodfellow and Badge, 1993). The non-invasive method of obtaining the foetal DNA from the maternal plasma was developed in order to minimise the risk of invasive methods. The expression of the male gene can be identified by Polymerase Chain Reaction by using a specific set of primers for the Y chromosome (Goodfellow and Badge, 1993).
The advancement of PCR has emerged as a powerful technique which is widely accepted in determining the gene expression. The PCR is a method for amplifying DNA. The basic principle involves the denaturation of DNA, primer annealing to the template and extension of the DNA with the help of the enzyme Taq polymerase. The DNA amplification can be calculated by the equation:
X=Initial amount of target, E= efficiency of amplification, n= number of PCR cycles.
It is possible to amplify over 30 million copies of DNA. PCR can be widely applied for the detection of pathogens, cancer cells etc.
The aim of the experiment is to determine the sex of the 3 unknown samples using Polymerase Chain Reaction and to determine the purity of the given Unknown 4 DNA samples using spectrophotometric analysis.
Materials and Methods
DNA purification from Tissue.
At first the water bath is set at 700 C and 560 C and the tissue from which the DNA is to be isolated was homogenised and transferred into 1.5ml eppendorf tube and 180Î¼l of ATL buffer was added to it.
20Î¼l of proteinase K was added to the eppendorf to digest the proteins and to inactivate the nucleases which might degrade the DNA if present. This was then incubated at 560 C with occasional shaking until the tissue was completely lysed.
20Î¼l of AL buffer was added to the sample and vortexed for 15 seconds and incubated at 700C for 10mins.
200Î¼l of 100% ethanol was added to the mixture and vortexed for 15 seconds. The lysate was then transferred into the Q1Amp Mini spin column. The column was then centrifuged at 13,000rpm for 1 minute. If the solution does not pass through the column this process is repeated. The column is placed into new collection tube and then filtrate is discarded.
500Î¼l of AW1 buffer was added to the column and centrifuged again at 8000rpm for 1 minute. The column was transferred into a new 2ml collection tube and the tube containing the filtrate was discarded. A 500Î¼l of AW2 buffer was added to the column and centrifuged at 13,000rpm for 3 minutes.
The filtrate was discarded and the column was placed back into the tube. The column containing the empty tube was centrifuged at 13,000rpm for 1 minute. The column was again transferred into a new 1.5ml and 200Î¼l of AE buffer was added to the column. The tube was incubated at room temperature for a minute and then centrifuged at 8000rpm for 1 minute. This step was repeated for one more time and then the column was discarded and the tube containing the solution was used for PCR (Spikings et al., 2010).
Polymerise Chain Reaction
The reaction mixture (Master Mix) was prepared in a tube containing all the necessary ingredients for PCR reaction; Buffers, primers, dNTPs, water and the enzyme Taq DNA Polymerase. (The PCR buffer contains the dNTPs, Taq Polymerase, and Mg2+). And the primer mix contains forward and reverse primers for SRY gene and GAPDH.
The known quantity of the master mix was then added into the tubes for control and unknown samples. The DNA samples were then added to all the reaction tubes and the final volume was made upto 20Î¼l.
A six 0.2ml micro-centrifuge tubes were labelled as follows.
"-", "P", "N", "1", "2", and "3".
The Master Mix tube labelled "-" was prepared according to the table.
Primer Mix (contain SRY gene and GAPDH)
12Î¼l of the prepared Master Mix was then added to the remaining 5 tubes. To these tubes 12Î¼l of the DNA sample was added.
Master Mix Volume(Î¼l)
Final tube volume
Unknown 3 (Isolated from tissue)
The tubes were then placed in the thermocycler for DNA amplification and the tube positions are noted down.
The PCR was programmed for a initial denaturation for 950C for 5 minutes.
40 cycles of
- denaturation at 940C for 30seconds
- Annealing at 600C for 30seconds
- Extension at 720C for 1 minute.
Final extension step was held at 720C for 10minute. The temperature of the cycler was held at 40C and the lid temperature was set at 1050C to prevent the tubes contents from getting evaporated. Later the samples were stored at -200C for further use (Spikings et al., 2010).
The primer used in the experiment is as follows.
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)
Agarose Gel Electrophoresis.
The DNA fragments were separated by electrophoresis. The 8 sample comb was placed in the mini-gel electrophoresis unit. The prepared gel was poured into the system upto 5mm depth and was allowed to settle for 15minutes.
A 4Î¼l of the sample loading buffer was added to each of the 20Î¼l of DNA present in 0.2ml tube. Once the gel is set, the comb was removed and electrophoresis buffer (TAE) was added so as to cover the gel by 0.5cm. From the tubes labelled "-", "P", "N", "1", "2" and "3" 20Î¼l of the sample was added into individual wells. And a ladder DNA of 4Î¼l was added to the very first lane. The gel was run at 100v for about 40minutes, until the dye runs half the way down the gel. Later the gel was viewed under the UV light and the image was stored (Spikings et al., 2010).
DNA determination is important when there is need to find the concentration or the amount of DNA present in any mixture. This is important when the DNA is isolated from any bacteria or any other organism. The DNA determination can be done by various means like either by reaction with chemical reagents or by spectrophotometrically. The most common method used is the spectrophotometer where in the DNA absorbs UV light at wave length of 260nm.
For the analysis, 1ml of water was taken in a cuvette and then placed in the spectrophotometer and blank was set by pressing "ZERO".
For DNA determination 20Î¼l of the unknown 4 DNA sample (4a, 4b and 4c) and 980Î¼l of distilled water was added into the cuvette and the absorbance was noted down at 260nm and 280nm. At 280nm the protein contamination in DNA sample could be identified. The ratio between 260nm and 280nm gives us the purity of the sample (DNA) (Spikings et al., 2010).
The Agarose electrophoresis gel was performed to separate the DNA fragments and to identify if the Sex Determining Region Y gene has been expressed. All the six amplified samples were subjected to Electrophoresis.
The gel results shows the expression of the House Keeping Genes (GAPDH) in the Positive, Negative, and in Unknown sample 1. Comparing the bands with the ladder DNA it can be said that the House Keeping genes are 200bp. Since the house keeping genes are involved in maintaining the basal cellular function they are present in all the DNA samples.
The SRY gene in the gel has not shown any expression. From the reference image we can say that the SRY gene is about 600bp in length. Comparing the bands with the ladder DNA shows that there is no band at the 600bp region. The Positive sample also has not shown any expression of SRY gene. In the Unknown sample 1 there is only expression of the GAPDH gene and not the SRY.
The Unknown sample 2 and Unknown sample 3 cannot be seen in the clearly in the image.
The reason for SRY gene being not expressed may be due to
The inappropriate loading of the DNA sample in the gel.
Contamination of the sample with other chemicals or substances.
The non-specific binding of the primer to the other template DNA.
PCR parameters may also be one of the reasons for improper expression of bands.
2 1 N P â€’ M
Reference Gel Image
Results of DNA Determination
The DNA determination experiment was done to find the purity of the sample. The DNA determination of the unknown 4 sample shows the concentration of the DNA present in 1ml mixture. The spectrophotometric analysis of the DNA sample is done at 260nm and 280nm. The analysis done at 260nm tells the absorbance of the DNA sample. Since the DNA samples are very much prone to contamination with protein and other organic compounds. And at 280nm it is possible to measure the protein contamination, since the protein absorbs light at 280nm. The ratio between the absorbance at 260nm/280nm for a pure DNA sample is 1.8. Our results have been compared with the standard ratio to measure the purity of the sample.
DNA concentration in 1ml (Î¼g DNA ml)
The dilution factor for the sample
DNA concentration in the 20Î¼l sample (Î¼g DNA ml)
The amount of DNA in 20Î¼l sample (Î¼g)
50Î¼g of DNA/ml or 40Î¼g of RNA/ml or 40Î¼g of single stranded DNA at 260nm gives an absorbance of 1.0.
Concentration of DNA in 1ml = 50 x Absorbance at 260nm
Absorbance at 260nm for 50Î¼g of DNA.
Concentration of DNA in 20Î¼l = Î¼g of DNA in 1000Î¼l x 20 x Dilution Factor.
Amount of DNA in 20Î¼l = Î¼g of DNA in 1000Î¼l x 20
The ratio of the Abs260/ Abs280 of all the 3 unknown 4 DNA samples indicate that the samples are not pure. In order for a sample to be pure the absorbance ratio should be equal to 1.8.
The introduction of the Polymerase Chain Reaction has enabled genetic determination of the sex at the embryo state in less than 72hrs. But many times due to contamination in buffers, DNA samples, enzymes, reagents interfere with the amplification process and hinder the accurate genetic diagnosis. The samples which have an intense Y band are recognised as males and the ones which have faint or no band are recognised as females. Our results show no expression of the SRY gene in any of the tested samples including the Positive control. The mis-diagnosis or inaccurate analysis may cause problems in judging the sex of the foetus by either producing a false band or absence of male specific band (Strom et al., 2001).
Since the electrophoresis results have no band relating to the Y gene in Positive control and Unknown samples 1, 2 and 3, there arises a doubt of inaccurate results. Comparing the gel image with the reference image it can be noted that the reference image has Y specific genes expressed in the Unknown sample 1. A further analysis of the PCR is required to confirm the results.
To overcome these problems there are certain techniques developed where there is simultaneous amplification of the Y- specific sequences and X-specific sequences. In this method the female is identified by X-specific amplification product where as the males are identified by the expression of both the X-specific and Y-specific bands. This technique helps in preventing the mis-diagnosis of the sex in pre-embryos in case of PCR failure. This method of gender determination was performed in 141 samples and none of them gave a false gender assessment (Strom et al., 2001).
Other experiments that were performed told that the sex determination done using the maternal DNA can be reliable only after the 7th week of the gestation. Quantitative analysis was performed to know the foetal DNA concentration in the maternal blood gene showed that the concentration of the foetal DNA in the maternal serum was 30.55copies/ml. This concentration gradually increased with progression of pregnancy (Honda et al., 2002).
From the DNA determination experiment it is possible to know the purity of the sample. The purity of the DNA sample is based on the amount of the DNA present in the solution and the ratio between absorbance at 260nm and 280nm.
For a PCR reaction the amount of DNA required varies from 5-100ng or 0.005Î¼g -0.1Î¼g. The yield of the PCR reaction depends upon the primer used for amplification.
Higher the primer concentration higher is the PCR amplification (Czerny 1996).
For a standard PCR protocol a 0.1Î¼M to 1Î¼M of primer concentration is sufficient. Experiments were performed by Czerny to check whether the ratio between the concentration of the primer and product has influence on the yield. Increasing the concentration of the primers from 1 to 20Î¼M showed that there was increase in the product yield upto 20 folds (Czerny 1996).
The spectrophotometric analysis of 4 DNA samples at 260nm and 280nm did not show a very good absorbance for all the 3 samples. The absorbance ratio at 260nm/280nm seems to be less convincing for the purity of the DNA.
Ratio for 4a: -0.167
The ratio for 4a is far close to the value of 1.8. It has a negative value which indicates that the sample is too contaminated with proteins or other chemicals.
The sample 4b seems to be comparatively better, although it is not in a very close range to 1.8.
Comparing all three samples we can say that unknown sample 4b is pure compared to 4a and 4c. To get a good PCR results the ratio of absorbance of the sample should be ~ 1.8. And hence none of the 4 DNA samples are suitable for a PCR reaction.
The two critical components that play a very important role in running a PCR successfully are: 1) the template DNA sequence, which should have a good quality and should not contain any inhibitors for Taq DNA polymerase. 2) Depends on the design of the oligonucleotide primer. The primer design mainly focuses on two aspects: specificity and efficiency of amplification. "Specificity is defined as the frequency with which a mis-priming event occurs" and "Efficiency is defined as how close the primer pair is able to amplify a product to the theoretical optimum of a two fold increase of a product for each PCR cycle" (Dieffenbach et al., 1993).
When designing a primer it is important that certain parameters are taken into consideration.
Primer length: The specificity usually depends on the length of the oligonucleotide primer and also on the annealing temperature. Primers length ranging from 18 to 24bp is said to be very sequence specific if the annealing temperature is set within a few degrees of primer temperatures (Tm). To optimise a PCR reaction it is better to have a primer of minimal length that has a melting temperature of 540C or even higher can have significant specificity and efficiency (Dieffenbach et al., 1993).
Terminal Nucleotide: The 3' end of the primer plays a very important role in controlling the mis-priming. The 3' end of the primer should not be complementary to each other, if not this will result in the primer-dimer phenomenon in which the PCR product will be the result of amplification between primers itself (Dieffenbach et al., 1993).
GC content and Temperature: The PCR primers should have base composition should be of 50-60% G+C content. At this percent the Tm of the primers will be around 56-620C which is the ideal temperature for efficient annealing.
PCR product length: It is possible to select a length for the PCR product. Generally the PCR product are of 150-1000bp in length. The size varies depending on the application. For a clinical trial 120-300bp length is sufficient where as for determining gene expression the length varies from 250-750bp (Dieffenbach et al., 1993).
The advancement in the bioinformatics field has made possible to develop various Primer design software and tools. One such program called MPprimer was developed for designing primers for Multiplex PCR. This employs the primer design program called Primer3 and also primer specificity evaluation software called MFEprimer which carefully evaluates the specificity of the primers and also check and prevents the primer dimerization (Shen et al., 2010).
Ideally, a primer should be at least 15bp in length and have 50% of the GC content in it. In our experiment the amount of GC content is less than 50% which might have affected the PCR product.
The melting temperature (Tm) also plays a role.
Tm= [40(% (G+C)) + 20(%( A+T))]
From this equation we can calculate whether there was any defect in the annealing temperatures.
Primer sequence use- SRY F - AAA CAG TGC AGT CGT ATG CTT CTG (24)
SRY R - GCC TTT GTT AGC GAG AGT AAG GAA (24)
For GADPH F- CCA ACG TGT CTG TTG TGG ATC TGA (24)
GADPH R - GAG CTT GAC AAA GTG GTC GTT GAG Â (24)
According to a study conducted in the Enzyme Application Laboratory, National Food Research Institute Japan, the annealing temperatures ranging from 420C to 900 C were experimented to get a good PCR yield. According to this experiment an annealing temperature of 500C for 30sec showed a faint amplified product. However when the temperature was decreased to 460C for 30sec, there was a sharp increase in the PCR product. When the temperature was decreased to 420C, there was positive PCR product but with many artefacts (Ahmed, 2006).
In our experiment we have used an annealing temperature of 600C for 30seconds. May be by decreasing the annealing temperature could have produced better PCR product.
The mammalian Y chromosome is said to be the main male determinant. The SRY's gene main function is to initiate the formation of the testis from the bi-potential gonads. This is done by triggering the differentiation of the sertoli cells from the existing pre-cursor cells. Sertoli cells are those which are associated with the germ cells and later develop into sperm cells.
This SRY gene codes for a protein called High Mobility Group domain (HMG) which has 78 amino acids. This SRY gene is referred to as the Testis Determining Factor (TDF) The mutation in the SRY gene and SOX9 (SRY related HMG Box gene 9) will result in 46, XY gonadal dysgenesis (Hanley et al., 2000). SOX9 is a transcriptional factor with a DNA binding region. The SOX9 regulates the fibroblast growth which is necessary for the growth of the sertoli cells. There are certain genes which are important in the formation of the bi-potential gonad.
They include Wilms' Tumor Supressor1, steroidogenic factor, empty-spiracles homeobox gene 2, and Lim homeobox gene (Wilhelm et al., 2007). These cells play an important role in the development of sertoli cells in males and granulosa cells in females.