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

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    Biology
  • Document type:
    Assignment
  • Level:
    High School
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DNA Extraction and PCR of bird DNA for sex identification

Introduction

Nowadays, there are different ways of collection and storage DNA. However, avoiding the nucleus impurities should be taken when handling DNA because it is may effects the DNA conservation.

Heterogametic term has been used in a study for determination between male and female in Pseudobagrus ussuriensis. Whereas, the male were heterogametic which mean they have two different chromosomes in there DNA XY while, the female have pairs of chromosomes and it is called homogametic. Richard (2004) claims that in the same study they observed that the length of the fragment for the DNA sample can be used to destinguch between male and female due to the male is longer then female. Moreover, in birds the female is heterogametic.

In accordance to Bruford (1998), monomorphic species mean there is no or little difference between male and female which make it hard to identify male from female in those species. In this study, different tissues from chickens have been used like chicken muscle, blood and feathers to identify the sex if it is male or female. Different tissues contain different amounts of DNA, which is why we tested different chicken tissues. For example, muscle tissues contains a lot more DNA than feather, and this will impact the results. However, this experiment done through several stages which will be explained in the next section.

The rationale behind the study was to determine the sex of the chicken, by using scientific techniques such as PCR and gel electrophoresis. By using PCR, the DNA extracted from the tissue can be amplified and then transferred to the agarose gel for gel electrophoresis. By using male and female controls in the gel, the sex of the chicken can be identified, as the Z and W DNA portions are different in length. If the sample only has one band it is a male, as the male sex is homogametic in males, whilst female chickens are heterogametic (ZW) and therefore two bands should be identified.

  1. DNA extraction and purification

A 20 mg of chicken muscle tissue placed into sterile 1.5 mL microcentrifuge tube then 180
µl of Buffer ATL mixed in the tube by using Vortex mixer for 15 second. Moreover, Vortex mixer have been used again after adding 20µl proteinase K for 15 second and once the tube have placed in a heat block at 56C for 30 minutes vortex for short time each 10 minutes to separate the sample. After this step 4µl RNase A mixed through vortexing for 15 second then the sample leaved for 2 min at room temperate. When those steps done the cells should be have lysed due to the breaking down of the cell membrane. Furthermore, the next step were vortex for 15 second then add 200
µl of buffer AL to the sample and again mixed for 15 second. Then added 200
µl of ethanol (96-100%, analytical grade) and again mixed by using the vortex mixer for 15 second. ‘The method of DNA extraction from other tissue types varied slightly. Full details are given in Hogan et. al. (2013).’

  1. Gel electrophoresis and PCR of DNA.

Gel electrophoresis is a process to separate DNA fragment. Firstly, preparing agarose gel. 1% agarose (w/v) in 1 X TAE buffer then placed in the microwave for one or two minutes to dissolve. After that allow the molten gel to cool to 50C in water bath. Secondly, adding the stock solution ethidium bromide to 150 mL of gel. The last step in the gel preparation is inserting the casting tray into the gel tank and place the black casting gates at each end then pour gel into it and finally insert comb which will create wells in the gel where the DNA sample will placed later on. The gel takes 30 minutes to set room temperature. Furthermore, in a clean microfuge tube added 10µl of genomic DNA with 2
µL of 6xloading- dye to get the sample ready for electrophoresis. After 30 minutes remove the black casting and the comb gel plates to add 1X TAE buffer slowly until it is covers all the gel by about 5mm. pipette 2-log ladder (1.0µg) and 5 µL of the molecular weight marker λ Hindlll in the first and last wells in the gel and places the DNA sample into an clean well. However, gel electrophoresis ready to be done after cover the electrophoresis and connect to power unit run at 180V for 40 minutes then turn off the power and remove the gel tray from the tank to plastic container.

  1. DNA visualisation on an agarose gel.

This is the last step in this report before getting the image and analysis the results of the DNA by using Gel doc apparatus to place the gel on the transilluminator box under white light. Notably Hebert (2004) urges that it is necessary to switch on the UV light and adjust the focus to make the gel and the wells obviously be seen and as well as adjust the exposure so that the marker and sample clearly to be seen. Finally, the image is ready to analysis the results

DNA Extraction and PCR of bird DNA for sex identificationDNA Extraction and PCR of bird DNA for sex identification 1

Figure 1 Feather tissue PCR Gel image.

DNA Extraction and PCR of bird DNA for sex identification 2

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DNA Extraction and PCR of bird DNA for sex identification 3

Figure 2 Muscle tissue PCR gel image

Tissue code

Non-drop concentration (ng/mh)

Final concentration required in 50µL reaction

Stock Solution Concentration

Volume of stock (solution added per 50µL reaction)

Number reactions per bench ( ___ reactions) + Controls (3) + Pipetting Error (1) = ____ reactions

1x PCR buffer

10x concentrated PCR buffer

200 µM dNTP’s

2mM each dNTP

1 U Taq DNA polymerase in 1x PCR buffer

2 pmol 2550F forward primer

2 pmol 2718R reverse primer

0.5mM MgCl2

2mM with PCR buffer Mg

Sterile milliQ water

26.5 µL (for 40 µL final)

Sample DNA or milliQ H2O

Discussion

From the electrophoresis diagram, you can see that the female control has two bright bands and the male control has only one bright band. We can also see the different bands of the molecular weight markers. Unfortunately, no one in my group had successful results, and this could be due to a number of reasons. We can also see from the diagram that the negative control had no bands. However, this doesn’t necessarily mean that the extraction of DNA from chicken muscle was unsuccessful, there could have been a whole range of possibilities for this to occur. One possibility could be that the pipette wasn’t working, another reason could be that all of the DNA didn’t leave the pipette when the gel was being loaded, or another possibility could be that the DNA wasn’t stored properly. It is unlikely that the gel electrophoresis apparatus wasn’t working properly on the day, because in all of the groups the molecular weight markers spread out across the gel, indicating that there was an electric current passing through the apparatus. DNA sample should be stored in a cold environment so it does not degrade and possibly my groups DNA sample was not stored correctly which why they did not show up in the gel. The DNA degradation was evident in muscle tissue because all of the group members were unsuccessful in producing bands on the gel. Probably this was because of incorrect DNA storage.

In relation with John S (1970), the pro of using the non-invasive technique like collecting chicken feathers is that it is really easy to find feathers in a chicken coop and also that you don’t have to hurt the chicken to get the feather. The disadvantage is that there isn’t much DNA in chicken feathers, so it would be difficult to determine the sex of the chicken. The advantages of destructive or invasive sampling is that these tissues like muscle and blood contain more DNA than feathers, but usually involve harming the chicken.

References

Cook, John S. Photoreactivation in animal cells. Univ. of Tennessee, Oak Ridge. Oak Ridge National Lab., Tenn., 1970.

Burke, T., and Michael William Bruford. «DNA fingerprinting in birds.» Nature327.6118 (1987): 149-152.

Griffiths, Richard, et al. «A DNA test to sex most birds.» Molecular ecology7.8 (1998): 1071-1075.

Hogan F, Loke S, and Sherman C (2013) SLE254 Genetics practical manual 2013. Deakin University.

Hebert, Paul DN, et al. «Identification of birds through DNA barcodes.» PLoS Biol 2.10 (2004): e312.