The DNA Mining and PCR of Bird DNA for Identification of Sex

It is extremely difficult to distinguish females and males in various bird species based on external characteristics. However, the difficulties have been met with the advent of molecular methods based on PCR. Currently, CHD gene primer amplification is the most dependable, cost-effective, and time-efficient process (Dubiec and Zagalska-Neubauer 2006, p.3). The consistency of DNA varies depending on the process of sampling, storage, and form of tissue used in the extraction. Since red blood cells contain nuclear DNA, DNA in birds is often obtained from blood. To obtain blood, the vein of the wing or leg is punctured and blood put in capillaries that are not heparinized. The site of blood collection depends on the species of the bird as well as the age. In addition, sampling can include collecting the DNA from the feathers, especially the ones that are growing. The basal angle of the calamus is the site whose cells are used in the extraction of DNA or from a clot of blood that is entrenched in the shaft (Morin, Messier and Woodruff 1994, p.994; Horváth et al. 2005, p.84). Moreover, a piece of tissue is usually sampled from an embryo, nestlings or dead adults, especially from liver or brain. It is important that all samples be stored under conditions that do not encourage degradation of DNA. DNA is commonly preserved in ethanol (96%), Queen’s lysis buffer or EDTA buffer. It is possible to store DNA in ethanol for many years because it is usually stable at room temperature. For more stability, DNA can be frozen at -80 or -20°C. Isolation of DNA from the sample is achieved through various methods that differ in terms of cost, effectiveness and labor extensiveness. Moreover, the selected techniques of sexing determine the extraction method that is most appropriate. There are different varieties of commercial kits that are available that enable the isolation of DNA that is of high quality (Hammond, Spanswick and Mawn 1996, p.299; Walsh, Metzger and Higuchi 1991, p.508).

It is not always easy to tell the difference between sexes in different taxonomic groups. There are birds that are dimorphic sexually, which can be easily distinguished between sexes. However, many different species have sexual monomorphism, thereby phenotypic characteristics that are similar, which make it hard to perform the identification of sex (Price and Birch 1996, p.842). Heterogametic sex denotes the species’ sex with dissimilar sex chromosome. For instance, human males have X and Y sex chromosomes. In contrast, in birds, the males are homogametic with ZZ chromosomes while females carry a copy of Z and another of W chromosomes (ZW) (Dubiec and Zagalska-Neubauer 2006, P.4). The establishment of approaches for molecular sexing was a breakthrough in sex identification in birds both in terms of rapidity and reliability. This cytological identification of sex is based on the morphology differences of the sex chromosomes (Solari 1993, p.43-73). Dubiec and Zagalska-Neubauer (2006, p.7) say that the most widely used tag for sex identification is offered by CHD gene and molecular markers have been designed to target the increasingly conserved primer flanking region within this gene. Therefore, the sex is determined through detection of size difference between CHD1W of the female and CHD1Z. DNA is extracted and the CHD1 genes are amplified by PCR before visualization through gel electrophoresis, such that one band represents a male (ZZ) while two bands represent a female (WZ).

The practical exercises were conducted to extract DNA samples from varying types of tissues including blood, muscle and feathers of a domestic chicken so as to be used in the determination of sex. Before the extracted DNA was used to amplify the locus that is sex specific, its concentration was determined as an assessment of both quality and quantity. Amplification applied the universally used sexing primers for avian, which included 2250F and 2718R, and which have been developed and used in the determination of sex of domestic chicken by Fridolfsson and Ellegren (1999, p.117).

Methods

Mining of DNA was achieved utilizing Qiagen DNeasy Kit for blood and tissue, which is a DNA purification kit that is commercially available. Using this kit, the cells of the sample are lysed by proteinase K and DNA is released into solution. The lysate put into DNeasy Mini spin column so that in the period of centrifugation, there is elective binding of DNA to the membrane. The contaminants that remain as well as enzyme inhibitors are washed off twice before pure DNA is eluted using water or buffer before its use. To purify genomic DNA from blood material that is tube for centrifugation (1.5mL). PBS (166 μL) was poured to the tube followed by RNase A (4μL) to degrade present RNA. The preserved blood card was cut and moved to the tube before it was incubated at 23°C for five minutes. AL buffer (200μL) was put and the content vortexed before incubating at 56°C for half an hour. 95% ethanol (200μL) was put to the sample afore mixing to precipitate the DNA. The spin column of the kit was taken and the code of the sample written on the lid before the liquid was put in using a pipette carefully not touching the membrane. It was then spun in the centrifuge at 8000rpm that lasted a minute before discarding the liquid that passed through as well as the tube used to collect the liquid. The column was placed in another tube (2mL) and AW1 buffer (500 μL) was put before it was centrifuged at 8000rpm to enable collection of liquid that pass through to be discarded. The washing was conducted again at 14,000rpm to ensure that the DNeasy membrane was dry. Another collection tube was added and centrifuged at 14,000rpm to remove any ethanol that could have remained. The mini spin column was put in a tube (1.5mL) that was labeled by the distinct code of the sample and the number of the group. Buffer AE (100μL) was put into the center of the membrane carefully not making contacts with the tip, and then kept warm for a minute at 23°C before centrifuging at 8000rpm to for elution. After discarding the column, DNA obtained was kept frozen. The approach of extracting DNA from feathers and muscle was a little different. Comprehensive details are provided by Hogan et al. (2017, p.25-31).

For visualization of the extracted DNA, gel electrophoresis was conducted. It works by separating the DNA molecules through electric current on a gel matrix. All the materials and reagents were gathered. Gel was prepared by 1% agarose in an electrophoresis buffer, which was boiled to dissolve before cooling it using a bath of water. To the gel, Ethidium bromide was put, which was later emptied into the casting tray before introducing the comb. Extracted DNA (10μl) as well as the loading dye was mixed. The casting plates were removed alongside the gel comb and the 1xTAE buffer was put to cover the entire gel. The markers for molecular weight and 2-log ladder followed in the holes of the gel. DNA was also put into the wells and positions recorded before running through power for an hour at 120V. Afterwards, imaging of the gel by Gel Doc system was done by assigning it on the transilluminator box. PCR was conducted on the extracted DNA and the CHD1W and CHD1Z results were viewed and analyzed. The process was similar to that of the previous electrophoresis except that PCR products were run.

Results

Section 1

Figure 1: Gel images of DNA from the varyig samples, which were blood, muscles and feathers. “B” denotes blood, ”M” denotes muscle and ”F” denotes feathers.

From the left of Figure 1, a DNA concentration/ size standard was included in the gel to serve as a molecular weight marker. DNA’s concentration was predicted by paralleling the brilliance of each of the DNA band to that of the corresponding ladder’s band.

Table1: estimated concentration of DNA from the three tissue types

Different DNA samples were collected from blood, muscle and feathers and their concentrations recorded as portrayed in Table 1 above. Different samples produced DNA of varying concentrations. M 17.06 had the highest concentration of 63.96ng among all muscle samples, B 17.25 had the highest concentration of 112.48 among all the blood samples, and F17.70 had a concentration of 84.37, which was the highest among all the feathers samples. The mean DNA concentration for feathers was found to be 15.24. The mean DNA concentration for blood samples was 61.73, while that for muscles samples was found to be 37.89.

From both Table 1 and Figure 1, it has been shown that the greatest concentration of DNA was derived from the blood sample, while the feathers had the least DNA concentration based on the means. From the gel images, it is clear that most of the feather DNA did not show any quality bands compared to DNA from blood and muscle samples. Table 1 also shows that majority of the DNA samples with low concentration were those extracted from the feathers. Blood tissue resulted in the highest yield of DNA and it is clear from Figure 1 that the bands are clearer compared to others.

Section 2

Figure 2: Gel image of amplified PCR materials from the different types of tissues, controls and the ladder.

As illustrated in Figure 2, it was easy to determine the sex of each of the samples that were shown on the gel. However, not all bands amplified. There was no negative control that amplified, which conformed to the expectation as there was no DNA contained in the negative control. The male and the female controls served as guides to help determine which samples were females and which samples were males. Male samples have only a single band, while samples were distinguished by the presence of two bands as shown from Figure 2. M17.03, M17.06, F 17.01, M17.01, B17.25, B17.36, M17.11, B17.33, M17.02, M17.10, M17.07, B17.27, B17.29, M19.09 and B17.35 were males, while there were no females detected may be due to the weak intensification of the female identification bands.

From Table 2, the tissue types amplified differently. Some DNA from the feather samples did not amplify, and those that amplified showed faded bands on the gel. The results that were obtained from PCR did not correlate with the actual sex of the individuals. From the positive controls that were used in this practical for both males and females, it was easy to tell the approximate base pair size of the W and the Z genes by comparing with the ladder. The W genetic factor is roughly 600bp, whereas Z protein sequence is nearly 800bp.

2.

Discussion

The results showed that DNA concentrations from the three types of tissues differed. DNA from blood was more concentrated than that from other tissues, while DNA from feathers had the least concentration. In the current practical, different sampling was conducted. Muscle was taken through destructive method, and blood was taken through invasive sampling, while the feathers were taken through non-invasive method. According to Harvey, Bonter, Stenzler and Lovette (2006, p.137), molecular sexing has less often employed the use of feather samples, although they are an interesting tissue type since plucking of feathers is a slightest offensive way of getting a genomic sample. When feather sampling is paralleled to specimen of blood, it entails a reduced amount of training, shorter handling time and it is less expensive. However, feathers have challenges as DNA sources. The feathers have less copy number of DNA that DNA obtained from tissue samples or blood. In addition, the feather DNA is to a certain extent degraded, especially if it came from cells that pass away. Based on the findings of this practical, the yields of DNA from feathers are correspondingly very low compared to DNA from other materials. Harvey, Bonter, Stenzler and Lovette (2006, p.137) say that DNA derived from feathers is a less dependable source of PCR template than genomic material from blood and tissues, which are richer tissue types.

On various feather-DNA samples, there were no visible PCR ensembles on the gel. However, it cannot be said that the extraction was ineffective. Samples collected through the non-invasive sampling like feathers have DNA that undergoes degradation over time, which may render then unreliable for genetic analysis. Feathers are not rich sources of DNA, and maybe the extracted DNA in the samples that failed to amplify might not have been sufficient, which is an observation that suggests that a bigger sample was required. Therefore, when choosing the sampling approach to apply, it is important to consider what is being done as it relates to the amount and quality of the required DNA.

It is very important to store various samples that are intended for analysis of the genome correctly because any impurities in the DNA results in inaccurate concentration measurement and has a potential to inhibit subsequent reactions. According to Arctander (1988, p.206), ethanol serves as an agent that kills and preserve in combination with cold so that DNA stays for several years. DNA quality is influenced by various aspects like preservation technique, preservation period, extraction method, and storage type and length. When DNA is incorrectly stored, it degrades by unfolding or breaking apart, thereby making any conclusions from its analysis invalid. It is important to safeguard DNA samples from any damage by proper methods of preservation, keeping the sample away from UV radiation, extreme temperature, pH and high salt concentrations. DNA of good quality has distinct bands that are bright and no smear is seen along the gel. In the current experiment, DNA degradation was evident in feather tissue as evidenced by the smears that were seen along the gel as shown in Figure 1, and the lack of PCR bands as evidenced in Figure 2.

DNA has to be sourced from an individual to allow analysis of its genetics (Freeland 2005, p.31). The three sampling methods include destructive, invasive and non-invasive. When enough DNA is needed for analysis, destructive sampling is employed, but its main limitation is that it involves killing the animal to get the required tissues for genetic analysis. Invasive sampling entails capturing the animal to get the biopsy or blood sample invasively and only a small sample is taken. This method is not destructive, and the main advantage is that only a small sample is required to extract sufficient DNA. In sampling that is not invasive, the DNA source is dropped and one can collect it not by grasping or distressing the animal, but the method is limited because the DNA obtained might have degraded and a big sample is required (Taberlet, Waits and Luikart 1999, p.324).

The experiment used universal primers to provide information regarding each individual’s sex. 2250Fand 2718R is the established widespread primers for sex identification in avian that were utilized to flank the fragment of the CHD gene so as to permit discernment between the PCR yields from the Z and W genetic material on the gel as indicated in Figure 2 (Dubiec and Zagalska-Neubauer 2006, p.8). Employing the universal molecular markers for sexing is advantageous because the amplification of the primers that are related to CHD gene has been shown to be the most cheap, rapid and reliable method of almost widespread use. Subsequently, females are identified by two bands, while the males are denoted by a single band on the gel. The CHD-based method is unquestionably the first technique of choice in molecular sexing as it is accurate and fast. Dubiec and Zagalska-Neubauer (2006, p.9) say that sample processing including extraction of DNA, PCR and its resolution on the gel takes approximately 5 hours or less. In addition, the appropriate CHD-linked primer pairs are chosen by checking those that have been applied successfully in species that are closely related.

This practical has provided proof that feathers afford insufficient DNA for reactions of sexing in birds by molecular method. The unswerving evaluation of feather against blood and muscle resulting DNA portray far-reaching inconsistency in identifying the sex of birds, which suggest that feathers as a source are connected to notably high PCR disappointment as well as rates of scoring error (Taberlet, Waits and Luikart 1999, p.324). In this practical, there were situations where the PCR band of the W gene, which is female specific was regularly pathetic compared to the band of the Z-chromosome, thereby requiring exceptional examination. As a result, the weaker intensification of the female identification band resulted in females being counted as males.

References

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Hogan, F., Loke, S. and Sherman, C., 2017. SLE254 Genetics practical manual 2017. Deakin University

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