Bird DNA Extraction and Polymerase Chain Reaction for Sex Identification

Introduction and Aims

Deoxyribonucleic acid (DNA) is a biological molecule that is used to encode genetic information. The information referred to in this case guides the development process and functions carried out by all living organisms. The data encoded in the DNA includes such elements as sex and traits of an individual. As the name indicates, the component is a form of a nucleic acid. It is made up of simple units that are referred to as nucleotides. The DNA molecules are found in the chromosomes, which are located inside the nucleus of the cell (Freeland 2005). Today, the study of the molecule is common. Scientists are currently conducting many experiments with the aim of effectively understanding this component.

In order to study DNA molecules, appropriate extraction methods should be applied. It is also important to note that the methods of collection and storage, as well as the tissue types used, have an impact on the quality and quantity of DNA obtained. There are several methods of DNA extraction. The strategies include ethanol precipitation, phenol–chloroform extraction, and minicolumn purification. Tissues from which the DNA is to be extracted also impact on the quality generated. The tissues should be fresh or frozen depending on their nature (Freeland 2005).

In the current study, DNA was extracted from muscles, blood, and feathers. Blood and muscles should be frozen. Feathers, on the other hand, can be kept at room temperature. Blood and muscles are rich sources of DNA (Taberlet, Waits & Luikart 1999). As a result, small quantities of the samples are required to generate genetic materials. The quality of the DNA from these sources is also high. Feathers are also good sources of DNA. However, the quantity provided is often very small. As a result, large amounts of samples are required. The quality of DNA extracted from feathers is also lower compared to that from other sources.

The sex of an individual depends on their sex chromosomes. The chromosomes vary between species. In birds, sex is determined by the Z and W chromosomes. Females and males have different genetic composition. To this end, the former are heterogametic. On their part, the latter are homogametic. What this means is that the females have both Z and W sex chromosomal components. Homogametic sex in males, on the other hand, means that they have 2 copies of a single chromosome. In this case, females are ZW, while males are ZZ. For example, domestic chicken are sexually mononophic birds (Freeland 2005).

Polymerase chain reaction (PCR) and gel electrophoresis have been used for purposes of molecular sexing in birds. The use of the two procedures is made possible by the discovery of CHD (chromo-helicase DNA) binding regions found to be both in the W and Z chromosomes (Vucicevic et al. 2013). Discoveries have been made with regards to primers that are CHD-specific. Such developments have helped in sex typing in these creatures.

Once the DNA has already been extracted and the required fragments amplified through the use of PCR, the primers are used for the purposes of showing fragments of the W and the Z sex bands that are clearly resolved (Ong & Vellayan 2008). One of the primer sets that are commonly used in this process is the 2250F/2718R. The primer set helps in sex determination in birds by producing a single fragment. However, the size of the fragment varies between males and females (Ong & Vellayan 2008).

The rationale for this study is to identify the sex of domestic chicken by analysing their DNA components. The study will seek to identify different tissues that can be used as the sources of the DNA (Horvath et al. 2005). The sources to be used in this case will include blood, muscles, and feathers. The quality of the DNA produced from each of the sources listed above will also be determined. In order to analyse DNA, the extraction process must first be finalised.

In light of this, the extraction method will be discussed (Horvath et al. 2005). The required fragments of the DNA must be amplified. To this end, the PCR process will be used to achieve the objective. The use of CHD- specific primers will be discussed in the section below. Gel electrophoresis will be used for the analysis of the fragments generated from the sex bands (Fridolfsson & Ellegren 1999). The reason is that the 2250F/2718R primer sets had been used. The results will be interpreted as per the primer used.

DNA Extraction and PCR for Sex Identification: Methods Used

A number of steps were followed to extract the DNA used in determining the sex of the bird specimens. The first step involved the collection of the tissues that were to be used for the study. To this end, a number of tissues were gathered in preparation for the research. Feather samples, blood, and muscle tissues were collected from the birds. A total of 22 samples were collected and labelled appropriately.

Appropriate storage methods were used to ensure that the genetic matter provided was not degraded prior to the commencement of the experiments (Hogan, Loke & Sherman 2013). It is noted that degradation of the specimens would have affected the validity of the end results. Extraction of the DNA was done using the Wizard® Genomic DNA Purification Kit (Cat. No. A1125, Promega, Madison, USA).

The decision to use the kit was informed by various factors. One of them was the fact that the instrument was found to be more effective compared to other chemical and mechanical extraction procedures. The methodology is associated with a number of advantages. For example, it helps in the standardisation of the procedures used in the extraction of samples and specimens. In addition, errors are reduced through the use of the kit (Hogan et al. 2013).

Blood samples were the first to have their DNA extracted. The method varied slightly depending on the sample used. For example, the procedure used on muscles differed slightly with the one used on the blood. Following the extraction of the DNA, the matter was quantified and qualified through the use of spectrophotometer. The 260nm/280nm standard was used. To avoid bias in the experiment, the DNA extracted from all the samples was then standardised. Standardisation was done by changing the concentration of DNA to 0.1µg/µl. The samples were then visualised through gel electrophoreses conducted in 1% agarose gel. The technique was used to determine the intensity of bands contained in the DNA samples collected (Ong & Vellayan 2008).

After the DNA was generated, the desired fragments needed to be amplified. The PCR reaction was used to achieve this objective. The CHD gene was the target region for amplification. The 2250F/2718R primer set was used during the reaction since they are specific to the CHD gene (Hogan et al. 2013). The two primers also had the same melting point making them ideal for the experiment which involved rapid heating and cooling.

The Mastercycler Gradient thermocycler was used to conduct PCR. The reaction started off with initial denaturation process which proceeded for five minutes at 95⁰C. 30 repeat cycles of the denaturation process then followed at the same temperatures for a period of 1.5 minutes. Annealing was done at 41.5^oC for 1.5 minutes. The extension process followed shortly after at 72⁰C for 1.5 minutes. Final extension was done at 72⁰C for 3 minutes.

The PCR products were then separated and visualised separately to determine the sex of the bird from which each of the samples was obtained from. In this case, 3% agarose gel was used for the electrophoresis. The compound was treated with ethidium bromide. The aim of this was to increase the visibility of the strands. Electrophoresis was done at 90 volts for duration of one hour. Visualisation was done under UV illumination and photographed as the results for the study.

Results and Findings

Three types of samples were used for the purposes of the current study. The samples were obtained from feather, blood, and muscle tissues of the birds. Blood components provided the largest quantities of genetic matter compared to the other specimens. Small quantities of blood were in this case required in order to produce the DNA. Muscle tissues produced higher quantities of DNA compared to the feathers. The findings to this effect are as indicated in table 1 below. The quantity produced by muscles, however, did not match that obtained from the blood samples. As a result, the amount of sample to be used for the extraction was fairly small.

From the table below, it is apparent that feathers produced the smallest quantity of DNA compared to blood and muscles. Large samples were needed to produce the DNA required for the study. Regardless of this, it is important to note that the quantity of DNA produced varies even between samples obtained from the same tissue type. The quality of the genetic component produced also varied across the different tissue types used in this research.

In line with this, it was found that muscle samples produced the best quality DNA compared to the blood and feathers. The concentration of DNA produced from the muscle tissues was high compared to that generated from the other samples (Vucicevic et al. 2013). To this end, some the muscle samples recorded a concentration that was as high as 51.9%. The findings are illustrated in figure 1 below.

Blood also produced DNA of high quality. The concentration of DNA extracted from the Blood samples was higher compared to that produced using feathers. However, the concentration was slightly lower compared to that obtained from muscles. Feathers had the lowest quality of DNA compared to the other tissue types used as samples. The concentration of the genetic material generated from the feathers in some samples was as low as 0.2%. However, the concentration of genetic material extracted differed even among samples from the same type of tissue. The table below shows the concentration of DNA in the three tissue types used:

Table 1: Concentration of DNA extracted from feathers, blood, and muscle samples of the bird.

From the table above, one is able to determine the mean concentration of samples from the three different types of tissues used. The mean is obtained by dividing the total concentration for the tissue type with the number of samples obtained from it.

1. The mean Concentration for blood samples was 123.5/7= 17.6429
2. The mean Concentration for muscle samples was 162.8/6= 27.1333
3. The mean Concentration for feather samples was 31.7/6= 5.2833

Table 2: A representation of sample ID and extraction.

The table above shows the results for the 22 samples that were tested. The sample ID helps us determine the tissue type from which each of the samples was collected. The for example, the letter ‘F’ in the sample ID ‘thur_am_f_tm&pb’ signifies that the sample was obtained from a feather tissue. Letter ‘B’ at the same position stands for blood samples while ‘M’ represents muscle samples.

Gel electrophoresis was used to determine the sex of the domestic chicken from which the samples had been selected. The determination of sex is made possible by analysing the base pair size of the Z and the W genes. Domestic chicken can either be ZZ or ZW. Males are ZZ while ZW are female. The size of the W gene is 122 bp. It can also be detected at 218bp in gene electrophoresis. The Z gene on the other hand can is viewed at 261bp and 231bp.

Discussion and Conclusion

Samples from the muscles generated higher concentrations of genetic matter compared to the others. The average concentration of DNA from muscle samples was 27.1333 while that of blood and feather was 17.6429 and 5.2833 respectively. The results were consistent with those that have been observed in similar studies. As such, researchers wishing to conduct a similar study in the future would be encouraged to avoid using feathers as a result of their poor quality DNA (Horvath et al. 2005). The amount generated is also small. Blood, on the other hand, is a rich source of DNA compared to muscles and feathers. However, the concentration of the DNA in blood is lower to that found in muscles a factor that lowers its quality. Muscle tissues are therefore the preferred source of DNA.

Gel electrophoresis was used for the purpose of visualising the sex bands. The presence of these bands was an evidence of successful extraction of the DNA fragments required (Fridolfsson & Ellegren 1999). The absence of a band would mean that the sex determining gene was not detected. In the event that the region to be amplified in this case the CHD gene was not present in the DNA extracted, no band would also have been visualised. The reason for this could be degradation of DNA due to improper storage of the tissues. Failure to stain adequately can also make the band to be invisible.

The quality of the DNA used for the purpose of genetic analysis depends greatly on the quality of the samples used (Freeland 2005). The samples should also be stored appropriately in order to avoid the degradation of DNA contained in them. The nature of the sample to be used dictates the method to be used for the purpose of storage. Of the three types of samples used as sources of DNA in the study, feathers are the easiest to store.

Feathers are also not likely to go bad easily unlike muscle tissues and blood that are easily degraded at room temperatures leading to the loss of physical and chemical properties. Refrigeration is used for the purpose of storing blood and muscle tissues at temperatures of -20⁰C. Since the samples may require being stored for long durations of time, refrigeration cannot be sufficient. Such tissues can therefore be preserved in liquid nitrogen at -196⁰C. Incorrect storage of the samples leads to degradation of the DNA contained in them through lysis. The idea behind freezing is to deactivate DNAses (the enzyme responsible for the degradation of DNA).

Up on thawing, it is important that the unused sample be refrigerated immediately to avoid degradation. There were cases of DNA degradation in the study. It was evident in all the tissue types. The conclusion has been arrived at following the fact that there is a great variation in the concentration of DNA obtained from similar tissue types. The samples with low concentration of DNA are likely to have undergone degradation of the nucleic acid as a result of improper storage.

Apart from storage related issues, there are a number of factors that make a particular tissue more suitable for the study than the other. Blood is a good source of DNA. It produces a good quality DNA. Blood is also rich in DNA. As a result, only a small amount is required. The amount of blood required in the study is relatively small and therefore does not in any way negatively affect the health of the domestic chicken used in the study. The use of blood as the sample for the experiment however is to some extent invasive since it requires the puncturing of blood vessels. In most cases, the blood is obtained after clipping off a small piece of a bird’s toe using a sterile cutting object.

The cut can result to an infection arising toe is in contact with the ground which harbours a large number of disease causing micro organisms. Feathers are good sources of DNA too (Horvath et al. 2005). Only a single feather will be required from the birds. As a result, there will be no great impact on the life of the domestic chicken. Unlike the use of muscle tissues, the use of feather is less invasive and does not result in much damage of body tissues. They are also easily stored at room temperatures without resulting to the degradation of the DNA. However, feathers are poor sources of DNA. The DNA obtained is also of poor quality compared to that obtained from the blood and muscle tissues.

Plucking of feathers can also lead to injuries on the skin of the bird. The punctured skin can easily be infected especially when the environment is infested with pathogens (Horvath et al. 2005). Muscles are also good sources of DNA. They quantity obtained is also high. As a result, only a small quantity of the sample will be required. However, the muscle tissues are expensive to store since they require refrigeration. The use of muscles as a sample is also invasive since it requires surgical procedures resulting to stress.

Primers are standard nucleic acid strands. They play an important role in DNA sexing since they help mark the starting point for DNA synthesis. The primers play an important role in the replication of DNA. The reason is that DNA polymerases only provide nucleotides to genetic materials that are already in existence. The PCR reaction likewise requires primers. The new fragment synthesised from the extension of the primer then becomes the template for another.

Primers are normally used in pairs. The second primer acts as the reverse of the first. The 2250F/2718R set of primers was used in this study. The use of the primers has been made possible by the fact that they are specific to CHD binding region present in the W and Z genes (Fridolfsson & Ellegren 1999). As a result, they lead to the amplification of the amplifications of the DNA fragments where the genes are located. The primers can then be detected through the use of gel electrophoresis since the lengths of the fragments viewed vary.

Genetic markers are important in the analysis of DNA (Ong & Vellayan 2008). The marker is normally used for the purpose of identifying an individual. A lot of experiments that involve an analysis of genetic information can be based on these markers. In this study, the CHD gene is our genetic marker. The gene is a universal sexing molecular marker in birds. It is present in both the Z and the W chromosomes.

The marker is suitable for molecular sexing since it is length in the two chromosomes vary. The reason behind this is the difference in the sizes of introns present in the two chromosomes (Ong & Vellayan 2008). PCR play a critical role in the determination of sex. To this end, they are used to amplify the CHD gene with the aid of primers. The process is made possible through the use of CHD specific primers, such as the 2250F/2718R set.

The PCR reaction is aimed to increase the number of the CHD genes (Ong & Vellayan 2008). However, this may not be possible in the event that the DNA has been degraded following the improper storage of the tissue samples. Poor quality DNA extracted from the tissues used may also impact on the PCR product.

DNA is one of the most important biological molecules. The molecule is responsible for all the traits in a living organism. For this reason, a lot of studies linked to the molecule have been carried out across the globe. Most of these studies are aimed at determining the sex of an individual. The current study was conducted to analyse the sex of domestic chicken by evaluating their DNA. The PCR reaction was important in the study since it helped in the amplification of the DNA fragments that were of interest to the study. The discovery of the CHD gene located in the Z and W sex chromosomes, a universal sexing marker, has made it easy for researchers to determine the sex of domestic chicken using universal premier sets.

References

Freeland, J 2005, Molecular ecology, Wiley, Chichester.

Fridolfsson, A & Ellegren, H 1999, ‘A simple and universal method for molecular sexing of non-ratite birds’, Journal of Avian Biology, vol. 30 no. 1, pp. 116-121.

Hogan, F, Loke, S & Sherman, C 2013, Genetics practical manual 2013, Free Press, New York.

Horvath, M, Martinez-Cruz, B, Negro, J, Kalmar, L & Goday, J 2005, ‘An overlooked DNA source for non-invasive genetic analysis in birds’, Journal of Avian Biology, vol. 36 no. 1, pp. 84-88.

Ong, A & Vellayan, S 2008, ‘An evaluation of CHD- specific primer sets for sex typing of birds from feathers’, Zoo Biology, vol. 27 no. 1, pp. 62-69.

Taberlet, P, Waits, L & Luikart, G 1999, ‘Noninvasive genetic sampling: look before you leap’, Trends in Ecology and Evolution, vol. 14 no. 1, pp. 323 – 327.

Vucicevic, 1, Stevanov-Pavlovic, M, Stevanovic, J, Bosnjak, J, Gajic, B, Aleksic, N & Stanimirovic, Z 2013, ‘Sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing’, Zoo Biology, vol. 32 no. 3, pp. 269-276.