SEX DETERMINATION USING POLYMERASE CHAIN REACTION

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About Authors:
Emanual Michael Patelia*, Rakesh Thakur, Jayesh Patel
Department of Pharmacology,
University of Bedfordshire, Luton, LU1 3JU, England.
*ricky.emanual@gmail.com

Abstract
The PCR is widely used technique in sex determination. PCR is also used for the determination of x linked inherited disorders, with help of biopsied embryos into mothers. DNA polymerase enzyme replicates a piece of DNA. Thus, chain reaction occurs and generating multiple copies of it. Polymerase chain reaction is frequently used technique for sex determination. PCR can also be used for detecting the presence or absence of a particular piece of DNA. In this method, we used PCR method for determination of the sex of three unknown bovine samples.PCR techniques have developed to reduce the problems by increasing amplification quality.

REFERENCE ID: PHARMATUTOR-ART-1911

Introduction:
Polymerase chain reaction is one type of the method used for the determination of the sex. Polymerase chain reaction method (PCR) was invented by Kary  B. Mullis, they was also received the Nobel prize in chemistry for PCR method invention on 10 December, 1993. [1] PCR Method also used in other detection like mutations detecting, monitoring cancer therapy, detecting bacterial, viral infection and also sex determination. [1]

Sex determination is one of the important methods, which help to determine the sex of the developing embryo. Mostly x-y system is use for the sex determination in human and mammals. Males have ‘x’ chromosome (xy) and female have both (xx) chromosome. So with the help of the chromosome sex  are determine. It is not true for all the organism, for example in bird and reptiles male have homogametic (ZZ) and female have heterogametic (ZW). Generally in male x and y chromosome and in female xx chromosome. sex is determined by the presence and absence of the y chromosome.[2]

We can determine sex with the help of presence of unique sequence on the y chromosome (SRY gene). The presence of SRY gene and protein encoded is very important for testis differentiation in the early embryo. if SRY gene is absent means development of ovaries. The protein of high mobility group (HMG) family is encoded with the help of SRY gene. After that this protein triggers all the step which is required to development of male phenotype.

Material and Methods:

DNA purification:
In the multiplex PCR method ,  In these unknown samples adding buffer AL ,BUFFER AW1 and buffer AW2 for isolation and purification of DNA from unknown sample. After addition to each given sample followed centrifugation.

Than transfer the column into new 1.5 ml tube than add buffer AE and incubated at room temperature and than centrifuge. After that discard the column and leave the tube on ice for PCR set up.

PCR:
In these PCR method we used different reagent for the PCR reaction like Buffer, primers,dNTPs, water and Taq DNA polymerase. First we marked the label on six 0.2 ml tube as a ‘M’, ‘F’, ‘1’, ‘2’ , ‘3’ and ‘-‘ .

Than we prepared a master mix in tube by added 15µl PCR buffer,dNTP mix 2.4 µl,primer mix 15 µl, sterile PCR water 103.2 µl,Taq DNA polymerase 2.4 µl . Finally, we prepared a 138µl master mix.

After that we aliquot 23 µl of the master mix into 5 remaining tubes and added 2 µl DNA to each tube. Than placed in the thermo cycler. In the last PCR thermal cycle keep for initial denaturation   at 95 °C for 1 min, annealing at 66 °C for 1 min, extension at 72°C and final extension at 72°C for 3 min. Than for remaining use stored the samples at -20°C.

Gel Electrophoresis:
First Place the 16 sample comb in the MIDI Gel electrophoresis. After that we poured gel solution (1.5% agarose) to a depth of 5mm and allow to set for 15 min.

Than we add 5 µl of sample loading buffer to each o.2ml tube from the PCR reaction.

When the gel has set. Take the comb and add electrophoresis buffer (TAE) to cover the gel by approximately 0.5cm.add 20 µl of all sample to individual well and than run the gel at 100v until the blue dye runs half way down the gel.

DNA quantification:
DNA quantification are help to determine the quantity of amplifiable DNA [3]. Quantities   analysis is a procedure where the concentration of a particular bimolecule in a sample is determined [4].with the help of a spectrophotometer the DNA and proteins are determine by absorbance of uv light. At 260nm absorbance DNA determined and 280nm absorbance protein contamination was estimated. The blank sample was used before measuring each sample absorbance.

Results:


Figure 1: A positive PCR reaction for SRY should have an amplicon of 741bp, GAPDH should be 218bp.

The first lane in the figure above is marker sample.
second lane is male
Third lane is female
Forth lane is sample 1
Fifth lane is sample 2
Six lane is unknown sample 3
Seven lane is negative control

In the second lane, it is for male control. Ideally two bands should be detected. One bands at the level of 218bp and second band at 741bp, First band for GAPDH gene and second band for SRY gene. In our experiment result two week bands are visible.

Third lane is for female. There is only one band are visible at 218 bp , which is for GAPDH gene. In female there is no SRY gene. In our experiment we detected week band at 218bp in second lane.

Forth lane is for unknown sample 1. This sample was female, there visible only one band at 218bp and there is no SRY gene visible. In our experiment one band is visible at 218 and no other band are visible.

Five lane is for unknown sample 2. This sample was male, there should two bands are visible, one band at 218bp for housekeeping GAPDH gene and second band at 741bp level for SRY gene. In our experiment one week band  at 218bp and no other band is visible.

Six lane is for unknown sample 3. This sample was male. there should two bands are visible, one band at 218bp for housekeeping GAPDH gene and second band at 741bp level for SRY gene. In our experiment two bands are visible; first band at 218bp for GAPDH and second week bands at 741bp for SRY gene.

Seven lane for negative control. There should be no band are visible. In our experiment bright band is shown, which is some similar to the primer dimer.

1. The terms regarding denaturing, annealing and extension refer to in PCR.
Denaturing means heating of the PCR products upto 94 °C for 30 sec .because of these hydrogen bonds between complementary bases is broken and get single stranded DNA molecules.

Annealing means lowering the temperature at 66°C for 1 min which are help in the binding of primers to single-stranded DNA templates.

Extension means  heating of mixture to 72°C for 1 min to which helps to DNA polymerase to synthesize a new DNA strand complementary to DNA template.

2. Explanation regarding the role of GAPDH primers in this experiment.
GAPDH genes are housekeeping genes.  GAPDH primers were provided to amplify these genes.

3. Explanation regarding the role of SRY primers in this experiment.
SRY gene which is sex determining region on Y Chromosome. For differentiation of testis in human embryo, the presence of SRY gene and the protein encoded by it are necessary. If SRY gene is absent, it results in development of ovaries SRY primers were provided to amplify these genes.

4. Calculation of GC content of each primer. Show your workings.
GC content= G+C/ A+T+G+C × 100

GAPDH F: 12/12+12 × 100 = 50

GAPDH R: 12/12+12 × 100 = 50

SRY F: 11/13+11 × 100 = 45.83

SRY R: 11/13+11 × 100 = 45.83

5. Calculation of the volume of each reagent in the master mix that will be present in each PCR tube. Show your working.
PCR buffer: 15/6 = 2.5

Dntp mix: 2.4/6 = 0.4

Primer mix: 15/ 6 = 2.5

Taq DNA polymerase: 2.4/6 = 0.4

6. Calculation for the final concentration of dNTPs present in each PCR tube. Show your workings.
C1V1 =C2V2

50 Mm  × 0.4 µL = C2 × 25 µL

C2 = 0.8 Mm

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