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DNA extraction and PCR of bird DNA for sex identification Essay Example

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Title: DNA extraction and PCR of bird DNA for sex identification

Introduction

DNA can be extracted from different types of tissues taken from an individual when doing genetic analysis (Medawar 1985, p89). For examples, blood and organs are good sources of DNA because a very small amount of sample is required to obtain large amounts of good quality DNA. The quality of DNA is influenced by the methods of collection, storage and tissue types. The quality of the samples used for the test influence the yield and quality of the DNA result. The fresh materials give the best results hence materials that are not used immediately should be stored at low temperature of -20oC or -70oC in a mixture of ethanol and dry ice. However, it is worth noting that repeated freezing and defrosting of stored samples will result to the precipitation of the DNA and reduced fragment size. Therefore, proper care should be observed when storing the sample materials (Semikhodskii 2007, pg121).

Heterogametic sex is the sex of a species possessing dissimilar sex chromosomes. In humans, the heterogametic sex is the male. The resultant gametes may contain either Y-chromosome or X-chromosome. However, this report focus on birds specie hence heterogametic sex is defined with regards bird species. In birds’ specie, the heterogametic sex in birds is the female (Wolfensohn &Lloyd 2007, pg27). They possess two dissimilar sex chromosomes namely: a W chromosome and a Z chromosome. It is often difficult to distinguish species from their phenotype given the fact that most of them are sexually monomorphic. The most appropriate way to determine the sex of these species is to detect the difference in size between the CHD1 on the Z sex chromosome and CHD1on the W sex chromosome. This is achievable through extraction of genomic DNA from a small tissue like feathers and blood tissues which helps in amplification of CHD1 genes using PCR. Apart from being cheap and high sensitive, the use of PCR to determine the genetic sex of birds is ideal since it only requires a small sample of material hence minimizing physical effects on the bird (Medawar 1985, p9).

This study was aimed at determining the sex of individual species using several samples DNA from blood, feather and muscle of Gallus gallus. During the process, an estimation of the DNA concentration was done and PCR was used to amplify CHD1 gene. The variation between the individuals was then visualized using gel electrophoresis. However, it is worth noting that this process is applicable to other closely related species because of the conserved nature of the DNA (Reinstein, 1999, pg77).

In order to determine the sex of a domestic chicken, the DNA from different tissue types of blood, feather and muscle was collected. The first part of the experiment comprises of the purification of the DNA samples using Qiagen DNeasy Blood & Tissue Kit. The amount and quality of DNA extracted varied between different types of tissues. Proper procedures were followed to ensure that the PCR is not inhabited by contaminants (Semikhodskii 2007, pg99). The flow chart below shows the purification procedure using Qiagen DNeasy Blood & Tissue Kit.

ample: Lyse Bind Wash EluteBy: 2By: 1By:S

From the flow chart, the first step was to lyse the cells of the tissue samples using the enzyme proteinase K. this released the DNA into solution. The second step was to load the lysate onto the DNeasy Mini spin column and Centrifuged for 1 minute a speed of 6000 x g (8000 rpm). The resultant liquid was discarded to avoid contamination. DNA become bound to the membrane as contaminant pass through during centrifugation. Remaining contaminants and enzymes inhibitors are eliminated in two wash steps after which the DNA was then eluted into eater or buffer, ready to use (Williams 2012, pg39). To elute DNA, the column underwent incubation for 1 minute at a room temperature followed by 1 minute centrifugation at a speed of 6000 x g (8000 rpm). The tube containing the DNA was then placed in the esky and all samples stored at a temperature of -20°C waiting for the next practical. One male (♂) control, one female (♀) control and one negative control are included. This is in addition to 10 μL of diluted DNA, water or neat DNA which were also included in the tube. The prepared PCR tubes was then placed into the thermo-cycler and a program optimized run to amplify the CHD1Z and CHD1W gene variants using a set of universal avian sexing primers 2250F and 2718R (Williams 2012, pg91).

Gel electrophoresis was the preferred method used to separate molecules on a gel matrix. This process included insertion of a comb into one end. Molten gel was then poured into a casting tray. The DNA was mixed with the loading dye after which the samples are loaded into the wells which were created by the combs. This was precedence by placement of the gel into the electrophoresis buffer. The loading dye is dense and as a result the DNA sinks into the well that contains colored indicator dyes. Closer examination of the electrophoresis process was undertaken. To increase the visibility of the DNA, a small amount of (0.5µg mL-1) of the chemical ethidium bromide was added into the gel (Williams 2012, pg111).

SECTION 1

By: 3Figure 1 DNA results

Figure 1 represent a copy of the gel showing DNA extracted from all three tissue types. The male sample has one band while the female sample has two bands. The sample number for this study was Blood B13.29 ♀.

Determination of DNA concentration

The concentration of DNA was calculated as indicated below

ng DNA in MWM reference band = ng/µL (Williams 2012, pg72)

µL of sample DNA

9.42 ng = 0.92 ng/µL

Table 1: Estimated concentration of the three tissues

M13.23 9.42ng

M13.31 23.13ng

M13.34 0.56ng

M13.32 6.5ng

B13.27 23.13ng

9.42ng x 2

9.42ng x 2

ng/µL 0.942 ng/µL

F13.24 0.56ng, 6.5ng

0.942 ng/µL

2.313 ng/µL

0.056 ng/µL

0.65 ng/µL

2.313 ng/µL

0.942 ng/µL,

0.942 ng/µL

0,942, ng/µL 0.942 ng/µL

0.56 ng/µL, 0.65 ng/µL

M13.22 0.65ng

0.56ng, 2.32ng

.56ng, 2.03ng

Control 0.56ng

.25 6.5ng, 6.5ng

B13.20 o.56ng

0.065 ng/µL

0.942 ng/µL

0.056 ng/µL, 0.0232 ng/µL

0.056 ng/µL, 0.203 ng/µL

0.056 ng/µL

0.056 ng/µL

0.65 ng/µL

0.056 ng/µL

2.313 ng/µL

Table 2: Mean DNA concentration for each tissue type

Blood sample

Muscle sample

Feather sample

1.023 ng/µL

0.864 ng/µL

0.04775 ng/µL

Figure 1 shows sex identification based on intronic length polymorphism on the gel. CHD1 gene was produced after performing PCR with 2550F/2718R. The results for the male and female samples differed in terms of one band and two bands respectively. Table 1 shows the concentration of DNA compared to the reference band while Table 2 shows combined results. According to Table 2, there exist differences in concentration of DNA for different tissues. That is, the concentrations of blood sample, muscle sample and feather sample are 1.023 ng/µL, 0.864 ng/µL and 0.04775 ng/µL respectively.

SECTION 2

By: 4Figure 2:

Figure 2 above is the gel showing the PCR product (for the three tissue types), positive controls (male and female), negative control and size marker. Column 1 contains size markers while columns 2-11 is representation of different single DNA extractions.

The total sample size was 20 with 11 being male while 9 being female. Samples from the blood and muscles showed higher amplification than samples from feathers. However, it is worth noting that the results obtained from the PCR are not positively correlated to the actual sex of the individual. For example, control sample in column 6 has two bands which show that it is female, but the reality is that the sample is male.

Discussion

Sex determining of animals in natural environment is important for a number of reasons such as understanding population structure and dynamics. DNA examination used as a tool for sex identification may be applied to biological samples of unknown sex, like preserved non-invasively collected specimens (Semikhodskii 2007, pg11). The results of the study lived to the expectation. Theoretically, there exists a difference in the concentration of DNA when extracted from different types of tissues. This argument is well demonstrated by the results which showed a higher concentration for the blood sample (1.023 ng/µL), followed by muscle sample (0.864 ng/µL) and lastly the feather sample (0.04775 ng/µL). The extraction process can be simply described as a success although there was some absence of visible band on the gel for a feather sample. This can be attributed to the size of the sample because small samples can contain DNA but not enough to be visible enough (Williams 2012, pg22).

The quality of the DNA results depends on the quality of the samples. Therefore, the samples have to be stored in a very conducive environment to avoid DNA degradation. In this study, high quality DNA results was ensured through storage of the samples at a temperature between 20oC or -70oC in frozen liquid nitrogen or a mixture of ethanol and ice. Greater caution should be taken to ensure that there was no repeated freezing as this may reduce the fragment size and precipitation of the DNA hence negatively affecting yield of DNA. The DNA might also experience chemical modification that results to shorter fragments upon purification hence decrease in the quality of the DNA results. However, despite the efforts put forward to ensure good storage of the sample, there might have been some lapse in the storage system and this also explains the absence of visible band on the gel for feather sample obtained (Semikhodskii 2007, pg56).

The universal primers, 2550F-2718R, amplified the regions of the sex-linked CHD-W and CHD-Z genes enabling the identification of polymorphisms and monomorphisms in the CHD-Z region. From the results, the male samples showed one band in agerose gel analysis as opposed to female sample that showed two bands. It is worth noting that DNA was observed after the addition of a small amount of 0.5µg mL-1 of ethidium bromide chemical to the agerose gel (Williams 2012, pg82).

The importance of using universal sexing molecular makers cannot be denied. Its work ranges from sex identification, parentage testing, genetic and infectious disease identification to marker assisted selection. Birds are like human being and can sometimes undergo trauma when physically affected. For this reason, the use of feather sample as opposed to blood to undertake as a source of DNA source was vital in limiting trauma that the bird might go through. Further appeal, DNA-based sex identification using feathers can be done without the help of a veterinarian since there are no anesthesia or surgery requirements (Wolfensohn &Lloyd 2007, pg45). In addition, this process of sex identification can be commonly done to any bird species. The study was undertaken at 99% confidence level and the information enhances the value of the bird.

References

Medawar P. B. (1985). Aristotle to zoos: a philosophical dictionary of biology. Cambridge, Harvard Univ Press.

Reinstein, R. (1999). Post conviction dna testing: recommendations for handling requests. [S.l.], Diane Publishing co.

Semikhodskii, A. (2007). Dealing with DNA evidence: a legal guide. London, Routledge-Cavendish.

WilliamS, T. D. (2012). Physiological adaptations for breeding in birds. Princeton, Princeton University Press.

Wolfensohn, S., &Lloyd, M. (2007). Handbook of Laboratory Animal Management and Welfare. Oxford, John Wiley & Sons. http://public.eblib.com/EBLPublic/PublicView.do?ptiID=238396.