University Genetics Lab Report: Coat Color Metamorphisms in Mammals

This genetics lab report details an experiment investigating coat color metamorphisms in mammals, specifically using a mouse model. The study aims to understand the role of melanin and the major genes/alleles influencing coat color. The methodology includes DNA extraction from blood, feather, and muscle samples, followed by PCR amplification of the CHD1 gene. The report describes the procedures for gel preparation, electrophoresis, and visualization of DNA fragments on an agarose gel. The results section discusses the relationship between migration rates and DNA fragment lengths, analyzing the data using a semi-logarithmic graph. The experiment also included procedures to determine the sex of the samples. The report highlights the importance of understanding genetic variation and its potential for predicting breeding patterns, emphasizing the application of molecular techniques like PCR in genetic research.

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INTRODUCTION
The studies of the characteristics of mammals have been the obvious way to reveal the way
genes cooperate in the determination of one character. In the genetic study, the mouse is
considered a good mammal. This is because of its size that is very small thus becoming easy to
maintain in the laboratory. Also the reproductive cycle of mouse is very short as compared to
other species(White et al 2012).The genetic determination of the specific characteristic may vary
from one animal to another(Vogel and Motulsky 2013). It is therefore very clear that mouse is
used just as a model. In most of the mammals like mice, there are several genes that interact to
determine the specific characteristic of the organism. For instance, understanding of the genetic
variation background is one of the crucial phenotypic traits that would assist in the identification
of the new genes. Some of these genes would have effects on the trait of the mammal(Tsoi et al
2015).
It is therefore very important to broaden our knowledge of the background of genetic
pigmentation. The results that are obtained may be used in the future prediction of the genetic
pattern of a breed. According to the present knowledge on the information regarding the
molecular basis, there is not yet a molecular test to be used in distinguishing the coat color types
in the available species. The experiment that has been conducted seeks to highlight the possible
molecular test that may be used for future predictions of genes for the trait(Tamura et al 2013).
Wild species are usually known to be uniform in their phenotypic characteristics. The species of
the organism show specific colors and patterns in their appearance. The extent of trait variation is
common among the domesticated animals. This preferably means that the domestic breeds are
the suitable models. The diversity of the characteristics results from the occurrences of the

biochemical activity of the melanocytes. These are the cells that are obtained from the ectoderm.
These cells are specialized melanin that are meant to protect the organism from the effects of the
UV radiations .The UV radiations normally have the potential to destroy the genetic makeup of
the organism and even lead to the skin cancer. This particular effect is common in the a rid and
semi-arid areas where the intensity of sunlight is very high.
Scientific methods have been used to investigate the possible means of predicting future
breeding patterns. Some of these method have been found reliable to some extent. The
polymerase chain reaction is a DNA technique of amplification that has seriously brought
changes to almost all the aspects of the research of the biology. Prior to the performance of the
PCR,there should be extraction of the DNA template from various biological sources.
Considering that the PCR is very sensitive, only a few copies of the samples of the DNA may be
required(Richards et al 2015).
It is important to note that the freshly extracted samples will give a perfect results as opposed to
the old DNA sample extracts. In order to have the specific sequence, the required primers are
designed in such a way that they correspond to the ends of the target. Such primers will hybridize
to the template of the DNA that will later mark the sequence to be copied
In order to perform the practice of PCR,there is mixing of the template of DNA and molar
excess of the primers. This is followed by the mixing of the four free deoxynucleotides and
other phases of the thermos table DNA polymerase. The experiment follows strictly those
requirements that are meant to generate perfect results. Use of hand gloves to avoid possible
contamination of the specimen is one such measures.
AIMS

The aim of the experiment will be to understand the role that is played by the melanin in coat
color metamorphisms in mammals while using a sample of mammals preferably a mouse. This
will be done to investigate and understand the characteristics of the major genes and their alleles
which are known to affect the nature and the organisms whose melanin are associated with the
coat color of the mammals. This will help in understanding of the basic relationship between the
basics that are controlled by these alleles of a gene and also how their operations can be
perceived in a phenotype.
In order to perform special examination of the genic effects including epistasis, there will be
need to pipette the tutorial of the user for experimental analysis. This will be done to extract
DNA from blood, feather and muscle. To visualize the extracted DNA products on the agarose
gel and perform the estimation process of the concentration of the extracted DNA, there will be
need to boost and amplify the CHD1 while using PCR.Interpretation and visualization of the
PCR product on the agarose gel will be done to check and analyze the gel so as to determine the
sex of the extracted sample.
MATERIALS AND METHODS
Practical one procedure
The sampled cards were picked. The examination of the picked cards was done while making
notes.
Practical two (A) procedure
Selection of the correct pipette for the volume and the pipette was set to the correct volume. The
tip was firmly pushed into the tip holder and this was followed by depressing the plunger gently

to the first stop. The tip was lowered while keeping the plunger in place. Slowly and with
control, the correct amount of the liquid was drawn. The liquid was dispensed through angle
variation. In order to remove the remaining solution on the tip, the plunger was depressed passed
the first stop to the second step. Finally the tip was depressed and button ejected to discard the
tip.
Practical two part (B) procedure
20uL of the proteinase was pipetted using 1.5mL tube. This was followed by addition of 166ul of
PBS in the tube was done .Addition of 4 uL RNase (100mg Ml) followed. One whole punch size
was cut using a sized sample of the preserved blood card of the chickens. This was followed by
incubation that was allowed for about 25minutes.Addition of 200 uL of Buffer was done.
Incubation was allowed at 56 degree Celsius. Addition of 200uL of ethanol (AR grade) to the
sample was performed. The DNA easy spin was then removed from the sterile bubble packing.
All the liquid from the tube was pipetted into the DNeasy column tube.
The spin column unit was then centrifuged at 6000X 8000rpm for 1min.The flow through the
liquid was discarded before the spin column unit was again centrifuged at 6000X 8000rpm for
1min.The DNeasy mini column was placed in 2mL collection tube and addition of 500uL
BufferAW2 done. It was then centrifuged for 3 mins (Okada et al 2013).Removal of the
DNAeasy mini spin column was done carefully so that the column could not come into contact
with flowing liquid. The collection tube was discarded and placed in the DN easy mini spin
column which was clean and with capacity of 1.5 mL.1oouL Buffer AE was directly pipetted
onto the DN esy center of the membrane. Incubation of the column at the room temperature for

1min was done. The column was discarded. Finally the tube with DNA was placed into the esky
at the front of the lab.
Protocol (b).
.A sterilized razor blade was used to macerate approximately 20mg of the tissue. Addition of 180
Ul of Buffer ATL to the tissue and vortex for 15s was then done. This was followed by addition
of 20uL of proteinase and mixed properly. The incubation was done for 30min at room
temperature of 56 degrees. Additional of 4uL RNase and incubation for 2mins was allowed.
Vortex for 15 seconds and addition of 200 uL Buffer AL was made. 200 uL ethanol was then
added. This was followed by removal of the DNeasy spin and subsequent collection of the tube
from the sterile bubble.
The mixture was pipetted into the DN easy column. The DNeasy minimspin column was
centrifuged at 6000Xg(8000 rpm).The minmispin was put into 2mL collection tube and then
added to BufferAW1.The minmispin was put into 2mL collection tube and then added to
BufferAW2.This was followed by removal of the DNeasy carefully to ensure that it did not
come into contact with another flowing liquid. The collection was discarded into the container
before addition of further 100uL Buffer AE to the solution. The column was discarded. Finally
the tube containing DNA sample was placed into the esky at the front of the lab.
Protocol (2C)
Apiece of feather was cut into sections using a sharp razor blade. Introduction of the piece of
feather to a sterile 1.5mL microfuge with 180 uL of Buffer ATL was done there was addition of
20uL proteinase K(Kumar, Stecher, and Tamura 2016)Allowed period of 15mins for vortex
stage was allocated before removal of the spin which was labeled with unique code. The mixture

was pipetted into the column and later allowed to centrifuge at 6000X8000rpm for 1min.The
spin was placed into 2mL tube and addition of 500Ul of Buffer AW2 done. Then centrifuge
process was allowed for 3min.The skin was carefully removed to ensure it did not come into
contact with the liquid flowing. The tube for the collection was discarded. Pipetting of 50uL
Buffer AE directly onto the membrane of the DNeasy was done before the column was
discarded. Finally the tube was placed with the DNA into the esky in front of the lab.
Gel preparation procedure
Preparation of 1% of agarose by boiling until it dissolved was done and then allowed to cool at
50 degrees was the first step. Addition of SYBR safely to the stock of 150mL of gel was done.
Finally a casting tray was put into the gel.
Electrophoresis
Careful removal of the black casting plates was the first step This was followed by pipetting of
5uL of the molecular weight .Pipetting the sample of the DNA into the well of the sample was
done followed by putting the cover on until the power was switched on. After the gel had run,
the power unit was turned off. This was followed by visualizing the gel using GEL Doc System.
Visualization procedure of DNAS was carried on an agarose gel. The gel was put on the
transillumonator. Adjustment to the focus under white light was done. Light was then switched
on. This was followed by improvement of the exposure through adjustment. Finally the image
was freezed and a copy kept.
Practical 3 Part (B) procedure

Preparation of one tube of the master mix was done. Boxes with the correct solutions were then
marked. To the matrix 0.2mL PCR was picked and added to 10uL with diluted DNA.The
symbols of male and females were finally used to do representation.
Practical 4 gel preparation procedure (Electrophoresis procedure)
Removal of the casting plates that were black was carefully done. This was followed by pipetting
5uL for generating molecular weight .The DNA sample was pipetted. Introduction of the cover
to the electrophoresis was then done. Once the gel had final running, the power was turned off.
Finally the visualization process was done using Gel Doc System.
RESULTS AND DISCUSSION
According the results that were obtained, the migration rates is inversely proportional to the
values of the logarithms of base ten of the toast band lenth.The plotted data produces the value
that has been indicated above. The line that had been produced allowed for the analysis of the
exponential ranges of the sizes of the fragments. It could be notice that the vertical axes of the
semi logs appear at the initial points but as the distance progresses there is shrinking effect that is
being realized on the graph. This has been the case because the scale representation has been
done using the logarithmic scale. The first scale that has been indicated on the y-axis corresponds
to the length that that is on the base pairs(Ffrench-Constant 2013).
The measurement ranges from as low as 100-1000 base pairs. The subsequent cycle measures
from 1000-10000 base pairs and so on. The major problem that occurred during the process of
amplification was the production of the unwanted products(Cruz 2013).

This was probably due to the contamination of the sample. Other than the effects of the
contamination, specific annealing of the primers was another reason that might have contributed
to such results. The exact temperature that was used during the process of incubation purely
relied on several factors. Some of the factors that were taken into the consideration included the
length of the DNA that was under target. Also the GC content of the template was a factor of
consideration(Ewens 2012).
Practical 2 part (A)
Q1. If there are 1000 μL in 1 mL, how many μL are there in 1L?
1000x1000=100000.
Q2 :0.225X1000=225
Q3: Q3. How many μL are there in 0.001 mL?
1000X0.001=1
Q4. If 1 ml of water weights 1 gram, then how much do the following weigh in grams:
- 500 μl
- 1000 μl
- 1 μl
- 1000 nil (nanowires)
500ul=500/1000=0.5g

1000ul/1000=1g
1/1000=0.0001g
1000x109x1000=1015g.
Questions for practical 2
Q1 What is the overall charge of the DNA
The DNA has an overall charge of negative(Ellinghaus et al 2013).
Q2 Why does the DNA move through agarosde gel?
The negative charge that exist on the phosphate of the sugar of the DNA normally force them to
move towards the positive electrode when they are in an electrical field.
Q3 The fragments of DNA migrate through the agarose gel based on their sizes.
Practical three Questions
Descibe how good quality and also high molecular weight appears when placed on agarosde gel
and compare the results with low quality fragments(Crowley, Di Nicolantonio, Loupakis, and
Bardelli 2013).
A good quality and high molecular weight looks like a perfect suspension with proper
inclination. They show very descriptive and bold structure that are visible. The low quality are
less visible.
Q2 Advantages of and disadvantages of non-invasive sampling

Advantages
It allows for the study of the wild animals in the parks without necessarily touching them.The
materials used in such cases include using shed air, shed feathers etc.
Disadvantages
The materials used like shed hair or the shed feathers are normally characterized by missing
information .This will leads to the genotypic errors mainly through allelic dropout(Carroll,
Grenier and Weatherbee 2013).
Advantages of destructive sampling
the destructive sampling DEALS WITH substances that are purely known.
Disadvantages
The result that is obtained will be different considering that the population keeps changing.
Q3 Which enzyme is responsible for the DNA replication? Helicase enzyme.
Questions for practical 4
Q1 What is meant by heterogametic sex and homogametic sex?
Heterogametic sex is normally that sex of a particular species in which the chromosomes of the
sex are not the same while the sex that have within their nuclei similar sex chromosome are
called homogametic sex.
Q2 Which sex is heterogametic?

Female ZZ
Q3List two reasons why scientists prefer using molecular markers
It is safe and reliable(Barrett et 2014).
It allows for the testing of a particular trait as early as the stage of embryo for the case of the
animals and also to access the embryo of the seeds before the seed are planted.
CONCLUSION
Mathematically, the expression of the PCR was done as an exponential relationship. After the
three rounds of the attempts of the experiment, it was found that there were eight copies of the
template of the DNA.If the process of the amplification could have been allowed for more than
20 cycles then the chain reaction would have produced more than even millions of the original
DNA templates.Theoritically this process would have continued indefinitely. The research found
PCR to be indispensible(Ammerman and Cavalli-Sforza 2014).
This was due to the fact that it was easy to use and its ability to have DNA amplification rapidly
done. The amplification processes of the PCR only needed a few materials for the start hence
making it ideal for the forensic analysis of the samples of the biology. Termination of the
paternity was also possible when this particular process was used. The comparison of the results
that was obtained with the already existing results confirmed that there were some relationships.
This basically means that the main aims of the experiments were met.