Introduction and Aims
DNA extraction in birds can be performed from different body parts such as feathers, muscles or other body tissues such as blood. DNA sample obtained from these different parts will however differ in quality and the best DNA sample is considered to be that obtained from blood. In this regard, only a little sample of blood is required for analysis such as VNTR. Despite this major advantage of DNA samples from blood, there is a major setback associated with such samples.
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Blood is more vulnerable to contamination especially when inappropriately stored. Blood also dries easily when exposed to air and this limits the amount of DNA that could be extracted. Muscle tissue is another good source of DNA samples. The process of extraction is quite demanding and could be destructive. DNA samples can as well be obtained from feathers but the quality of such samples is lower than that obtained from blood or muscle tissues.
When extracting DNA from feathers, the whole of the feather may not contain sample DNA. It is therefore important to consider extracting DNA from the feather tip or a blood spot located at the feather tip.
Organisms can either be heterogametic or homogametic. Organisms that have similar sex chromosomes are said to be homogametic while those that have different sex chromosomes are said to be heterogametic. Female human beings have XX sex chromosomes and therefore regarded as homogametic. On the other hand, males have XY sex chromosomes and are therefore regarded as heterogametic. In organisms of the class aves, things are different; males are homogametic as they contain ZZ sex chromosomes while females are heterogametic containing ZW sex chromosomes.
Birds unlike humans are monomorphic. This means that they cannot be differentiated by their phenotypic characteristics. This calls for use of sexing primers to detect differences in size between the Z and the W chromosomes and consequent visualization of isolated DNA by gel electrophoresis. Polymerase Chain Reaction (PCR) results from such analysis will show two clear bands to distinguish females as males’ results will show only one clear band.
The experiment mainly aimed at determining the sex of Gallus gallus through a practical process. To begin with, DNA extraction from the bird specimen was to be done. DNA was obtained from blood, muscle tissue and feathers of the bird. This was followed by estimation of DNA concentration and its consequent amplification of CHD1 using PCR. The last step was to visualize the DNA extract through gel electrophoresis and making conclusions of the bird’s sex.
Methods and Materials
Extraction and Visualization of DNA
A small portion of about 2-3mm of a feather tip was obtained from the bird. The portion was divided into several parts since we could not obtain a blood spot from the feather. These pieces were then sterilized by immersing them in a 1.5ml micro centrifuge tube containing 180µl ATL buffer. This was followed by vortex mixing for about 15s. 20µl of proteinase K was then added and the vortex ran for another 15s then incubation for 30 minutes at 560C. During this incubation, mixing in the vortex was done at intervals of 10 minutes. The sample was then lysed and mixed in the vortex before addition of 200µl of AL buffer then running the vortex for another 15 seconds. 200µl of absolute ethanol (96%-100%) was then added and the vortex ran for another 15s to obtain a homogeneous solution.
DNeasy spin column tube was removed from a sterile bubble and labeled. The micro centrifuge mixture was then transferred into the DNeasy unit and centrifuged at 8000rpm for a period of 1 minute. The column was then placed in a 2ml collection tube in which 500µl of AW2 buffer had been added. This was then centrifuged for 3 minutes at 17000rpm. This aided in drying the DNeasy membrane.
The DNeasy tube was emptied and the spin column and collection tube centrifuged for 1 minute at 17000rpm to dry the ethanol that could have remained. The DNeasy was transferred to a 1.5 ml micro centrifuge tube that contained 50µl of AE buffer then incubated for 3 minutes at room temperature. Following this was centrifuging at about 8000rpm for a minute. The DNA isolate was then refrigerated at -200C as the collection column was discarded. DNA isolation from muscle and blood tissue differed significantly.
Visualization of isolated DNA was done in agarose gel electrophoresis. The gel was prepared from 1% (w/v) agarose in 1x TAE buffer. This was boiled to dissolve then cooled to 500C. About 15µl of cyber café was used as the dye in place of ethidium bromide. The gel was given 30 minutes to settle before the gel comb was removed and 10µl of the isolated DNA mixed with 2µl of the loading dye. TAE buffer was then poured into the gel to cover it wholly. λ HindIII markers (5µl) were mounted.
On the first and the last wells, 2-log ladder was placed using a micropipette before the gel was run at 120V for 60 minutes. DNA is negatively charged and therefore runs from the cathode towards the anode. The gel was then placed in a plastic container where it was visualized with gel Doc system and the concentration determined.
PCR and Visualization of PCR products
Since more DNA was needed in the subsequent procedures, need for the DNA sample amplification was inevitable. This was achieved by using PCR. A master mix was prepared and the stock contained concentrations as shown below:
|Final concentration required||Volume of stock per 50µL|
|1x PCR buffer||5µL×8|
|1 U Taq DNA polymerase in 1x PCR buffer||1µL×8|
|2 pmol 2550F forward primer||1µL×8|
|2 pmol 2718R reverse primer||1µL×8|
|Sterile milliQ water||1µL×8|
|Sample DNA or milliQ H2O||1µL×8|
40µl of the master mix was then pipette into 0.2ml PCR tubes and 10 µl of the DNA isolate added. A male, a female and negative control were also run alongside just to be sure. A total of 7 tubes were prepared which included 4 reaction tubes and 3 controls.
The tubes were transferred to a thermocycler where amplification of CHD1Z and CDH1W was to take place with 2250F-2718R primers. The PCR products were then observed using gel electrophoresis in 1% agarose in 1x TB buffer. A similar procedure of gel preparation as described earlier was followed but SB buffer was used in place of TAE and the genetic marker used was 5µl of 100bp in length instead of of λ HindIII. A higher voltage to run the gel was required and therefore instead of 120V, a voltage of 300V was used for 20 minutes.