Reference no: EM132371606
Genetics and Genomics Assignment -
Practical 1 - Epistatic interaction between genes: the genetics of mammalian coat colours
There are no hazardous chemicals or equipment used in this practical.
Aims -
1. To understand the role of melanins in coat colour polymorphisms in mammals.
2. To investigate the major genes, and their alleles, that affect the nature and organisation of melanins associated with coat colour in mammals.
3. To understand the relationship between the primary control by alleles of a gene and how their action may be perceived in a phenotype.
4. To examine special genic effects such as epistasis.
Examine poster 1 related to cats.
Q1. What is the genotype, with respect to the 'S' gene locus, of the cat
a. with some white?
b. with extensive white?
Q2. Consider the actual action of the 'S' gene and offer an explanation for the fact that cats with the same genotype with respect to this gene can show a difference in the distribution of non-pigmented areas.
Q3. What is the genotype of the Blue Burmese cat with respect to the 'C' gene locus?
Q4. Explain which kitten has been miss-sexed.
Q5. What colour is the father of the litter? What colour is the mother of the litter?
Q6. A Siamese cat has an operation in the abdominal region. During this operation a patch of fur is shaved off. When the fur regrows, it is much darker than the fur in the surrounding area.
a. Suggest an explanation for this observation.
b. Explain whether you believe the patch of fur would remain dark permanently or lighten over time.
Examine poster 3-cats showing a mother cat with her litter of seven kittens.
Q7. a. What is the genotype of the mother at the 'B' gene locus?
b. What is the genotype of the mother at the 'C' gene locus?
c. What is the genotype of the mother at the 'S' gene locus?
Examine poster 3 showing a brown tabby cat.
Q8. What is the genotype of the brown tabby cat at the 'B' gene locus?
Examine poster 4 - cats.
Q9. What is the genotype, with respect to the 'B' and 'D' loci of a
a. blue point kitten
b. lavender point kitten
c. brown point kitten
Q10. a. What is the genotype of the blue cat at the 'B' and 'D' loci?
b. Is this identical to any of the kittens in the litter shown? If so, at which gene locus does it differ from the kittens to give such a difference in appearance of colour distribution?
Q11. You are provided with two dice that represent two parents of a particular genotype at the 'D' locus.
a. What is the genotype of each parent?
b. What phenotypes are possible in any kittens of these parents and what is the chance of each kind?
Generate six kittens.
c. Use the binomial expression to calculate the chance of these two parents having a litter of the same phenotypic make-up that you obtained.
Examine posters 5 and 5X.
Q12. Look at the photographs of Spadgie and Emily. These two cats have the same genotype at the 'O' locus.
a. On which chromosome is the 'O' locus?
b. What is their genotype with regard to this locus?
c. Explain why the distribution of black and orange fur is different in each cat in spite of the identical genotypes.
Q13. Note the photograph of the blue and cream tortie. In what way is the genotype of this cat
a. similar to that of the other torties shown?
b. different from that of the other torties shown?
Examine poster 5L that show the results of reciprocal crosses between black and orange cats. Neither of these crosses show orange females and yet orange females can and do exist.
Q14. What crosses would have a chance of producing orange females?
It is incorrectly claimed by some that orange female cats are not possible. However, they certainly appear less frequently than do orange male cats.
Q15 Write an explanation for this difference in sex frequency with orange cats that could be readily understood by a non-student of genetics.
Q16. What are the genotypes of the three tabby cats with respect to the 'B', 'T' and 'A' loci?
a. Tabby cat on the left-hand side of pair photograph.
b. Tabby cat on the right-hand side of pair photograph.
c. The Abyssinian tabby.
Q17. Explain whether you think the pair of tabby cats could be from the same litter.
Q18. What is the genotype of the black cat with respect to the 'B', 'T' and 'A' loci?
Q19. What is the term given to the genetic phenomenon that exists between the 'T' and 'A' genes?
Examine poster 7.
Note the agouti ticking at the ends of some of the hairs.
Q20. What changes in kind of pigment and distribution within hairs cause the agouti pattern?
Q22. Were the parents in each problem homozygous or heterozygous at the W locus? How do you know?
Q23. Analyse the data on both of the special problems poster. Use the information given to establish the genotype of the parents at the B, D, S & T loci, for each of the special problems.
Examine the phenotype of Bes Bes.
Q24. Predict her genotype at each of the following gene loci
S, C, B, D, O, T, A, I, W
Generate your answers in the form of a table with three columns with headings
Gene
Phenotype
Possible genotypes
ASSESSMENT - Work through the questions asked in relation to each station and make sure to take notes and complete all questions (including the special problems) related to the stations.
Practicals 2-4: Determining the sex of the domestic chicken (Gallus gallus)
Your Task - Your task is to extract DNA from one of three tissue types and use that DNA to determine the sex of domestic chicken (Gallus gallus). You will be allocated one of three tissue types for an individual - 1) feather, 2) muscle, or 3) preserved blood.
This experiment will be run over three practical sessions.
- Practical 2 DNA extraction
- Practical 3 PCR
- Practical 4 Gel electrophoresis
Aims
1. Pipette user's tutorial (Prac - 2, Part A).
2. Extract DNA from muscle, blood or feather of Gallus gallus (Prac - 2, Part B).
3. Visualise your extracted DNA product on an agarose gel and estimate the concentration of the extracted DNA (Prac - 3 (Part A)).
4. Amplify the CHD1 gene using PCR (Prac - 3 (Part B)).
5. Visualise your PCR product on an agarose gel (Prac - 4).
6. Analyse the gel to determine the sex of your sample (Prac - 4).
Assessment - 1 x scientific report in poster format.
Practical 2 (Part A) Pipette user's tutorial: an essential skill for molecular biology
Knowing how to pipette properly is an important skill in molecular biology. Pipetting errors are one of the most common reasons for experiments to fail in molecular biology classes (not reading the lab manual carefully would have to be the 1st ). Pipette volumes generally range from 10 mL down to 0.1 µL. Most often, the volumes you will need to dispense with pipettes will be less than 1 mL or 1000 µL (microlitres). It is helpful to start thinking of a µL as an everyday unit.
Q1. If there are 1000 µL in 1 mL, how many µL are there in 1L?
Q2. How many µL are there in 0.225 mL?
Q3. How many µL are there in 0.001 mL?
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 nl (nanolitres)
Practical 2 (Part B) Extraction and purification of DNA from different tissue types
Introduction - In this practical you will be using a commercial DNA purification kit (Qiagen DNeasy Blood & Tissue Kit) to purify DNA from chicken blood, muscle and feathers. The amount and quality of DNA extracted will often vary between different tissue types.
The DNA you extract in Practical 2 will be used in Practical 3 to perform a PCR (polymerase chain reaction). In order for your PCR to work you will need to be careful and follow the manufacturer's instructions closely. PCR can be inhibited by contaminants that are carried over from your DNA extraction if the procedure is not performed correctly.
The DNeasy Blood & Tissue procedures are outlined in the flowchart below. The cells of your tissue sample are first lysed (broken apart) using the enzyme proteinase K. This releases the DNA into solution. The lysate is then loaded onto the DNeasy Mini spin column. During centrifugation, DNA is selectively bound to the membrane as contaminants pass through. Remaining contaminants and enzyme inhibitors are removed in two wash steps and the DNA is then eluted into water or buffer, ready for use.
Each pair will be provided with one tissue type (blood, muscle or feather) to extract DNA from. Three protocols are provided in your notes. You will need to follow the appropriate DNA extraction protocol for your tissue type.
Important: Centrifugation steps need to be carried out at the correct speed. Check each step carefully.
Practical 2- Questions -
All questions need to be completed before leaving the practical class and this page needs to be signed by the demonstrator.
1. What charge does DNA have?
2. Why does the DNA migrate through the agarose gel?
3. DNA fragments migrate through the agarose gel based on their _______?
a. Weight
b. Fluorescence intensity
c. Size
d. Shape
Practical 3 (Part A) Gel electrophoresis of extracted DNA
Introduction - Gel electrophoresis is a technique used to separate DNA molecules on a gel matrix using an electric current. The gel is prepared by pouring molten gel into a casting tray with a comb inserted into one end. When the gel sets the comb is removed. The fingers of the comb leave behind small wells. The gel is then placed into an electrophoresis tank and the reservoir is filled with an electrophoresis buffer. Before loading samples into the wells the DNA is mixed with a loading dye. The loading dye is a dense solution which makes the DNA sink down into the well and contains coloured indicator dyes. The dyes travel at a predictable rate through the gel, enabling the progress of the electrophoresis to be monitored.
When the electric current is applied to the gel the negatively charged DNA molecules move through the gel matrix toward the positively charged anode (red cables) and away from the negative charge of the cathode (black cables). Smaller DNA molecules migrate through the gel (towards the anode) at a faster rate than larger DNA molecules.
DNA is visualised by the addition of a small amount (0.5µg mL-1) of the chemical Ethidium Bromide/ SYBR Safe to the gel. These stains intercalates with DNA molecules and fluoresces under UV light, therefore DNA can be visualised as fluorescent bands in each of the lanes in a UV transilluminator.
*NB: Ethidium Bromide/ SYBR Safe binds to DNA and therefore may be a potential carcinogen. Do not handle the gel.
The size and concentration of a DNA fragment can be estimated by including a DNA size / concentration standard, also called a molecular weight marker, in one of the wells. The size of the band is estimated by comparing the DNA band of your sample to a band of known size in the standard. The concentration of DNA can be predicted by matching the brightness of your DNA sample band to the brightness of a band in the standard.
Practical 3 (Part B) Polymerase Chain Reaction (PCR) of DNA
Introduction - Polymerase chain reaction is an enzyme-based method used to amplify fragments of DNA ranging from 100's to 1000's of base pairs in length. The reaction is performed by thermostable DNA polymerases. One of the most commonly used polymerases and one that we will use in this practical is called Taq polymerase. This enzyme was originally isolated from Thermus aquaticus, a thermophilic bacterium native to hot springs and thermal vents.
In order to copy specific regions of DNA, specially designed oligonucleotides (short pieces of DNA) called PCR primers are added to the reaction. These primers bind to complementary sequences on the template DNA at either end of the region of interest. The Taq polymerase then synthesises copies of the DNA by adding dideoxynucleotide triphosphates (dNTP's) to the end of each primer sequence. That way, both strands of DNA are duplicated.
Practical 3 - Questions
All questions need to be completed before leaving the practical class and this page needs to be signed by the demonstrator.
1. Describe how good quality, high molecular weight (HMW) DNA looks like on an agarose gel compared with low quality highly sheared (fragmented) DNA.
2. Give one pro and one con for the different types of sample collection; destructive sampling, invasive sampling and non-invasive sampling.
3. During the PCR reaction, which enzyme is responsible for replicating the DNA?
Practical 4 Gel electrophoresis and visualisation of PCR products
Introduction - In this practical you will view and analyse the results from the CHD1W /CHD1Z gene PCR you carried out in the previous practical session. Based on published data (Fridolfsson and Ellegren, 1999), we expect that the PCR fragments amplified with the 2550F-2718R primer will be smaller than 1000bp.
You could use a TAE, SB or TBE buffer to resolve fragments under 1000bp. The procedure will be essentially the same as the last electrophoresis practical except that you will be running PCR products rather than genomic DNA.
NOTE: The results for the entire prac class will be used for your scientific report and it is important that you record ALL the results for your prac class.
Practical 4 - Questions
All questions need to be completed before leaving the practical class and this page needs to be signed by the demonstrator.
1. What is meant by heterogametic sex and homogametic sex?
2. In birds, which sex is the heterogametic sex?
3. List two reasons when a scientist may want to use molecular markers to sex a (non-human) organism?
Practical report in poster format: DNA extraction and PCR of bird DNA for sex identification.
Your Report - The results from the DNA extraction practical and the PCR practical are to be written up as a single scientific poster presentation. You should include: Introduction & Aims, Methods (DO NOT copy the full protocol from your practical manual), Results, Discussion, Conclusion and References.
Please use PowerPoint to make your poster and make a new presentation with a single slide. From slide "layout" (under the home tab) choose a completely blank slide and adjust the slide size to poster dimensions (70 cm × 100 cm). You can make your poster in portrait or landscape orientation. You can then arrange text boxes and images on your slide as you so desire. A poster should be predominantly visual so you want to keep your writing to a minimum and use point form over full sentences. All elements on your poster should be visible from approximately 2 m away so don't use any fonts smaller than 16 point and make sure your images are large enough and clear (however if you use any extra references, these can be included in small print in a small box at the bottom of the page).
Report in a poster format should include:
Introduction - Break your introduction up into separate short sections that deal with a particular issue one at a time. Include:
- A section outlining how different methods of collection, storage and tissue types can result in varying quality of DNA.
- A section explaining what we mean by the heterogametic sex and how markers can be used to distinguish between males and females in monomorphic species or their young.
- At least 3 JOURNAL article references in the entire introduction section (this does NOT include websites, the prac manual or the unit guide, these are not valid references and should not be referred to).
- Introduction - Aims: Discuss rationale for your study.
Methods - Break this section up into sections like you have done in the introduction. Use dot points to give a summary of the methods used. Use the past tense. Include:
- A section on DNA extraction and visualization. Outline the DNA extraction method you used for YOUR sample only (please then include the following statement: 'The method of DNA extraction from other tissue types varied slightly. Full details are given in Hogan et. al. (2017).' Please include the following reference for the prac manual in your reference section: 47 Hogan F, Loke S, and Sherman C (2017) SLE254 Genetics practical manual 2017. Deakin University.
- A section on PCR and visualization
Results section - NOTE: The results for the entire prac class will be used for your scientific report and it is important that you record ALL the results for your prac class.
Include two sections, dealing with DNA extraction and PCR separately. You need to have the appropriate tables and figures for each section plus a few sentences describing the results and which refers to the table and figures. All tables and figures need to be referred to in the text.
Section 1
- A copy of the gel from your prac class showing DNA extracted from all three tissue types
- Table showing the estimated concentration of the three tissue types. Use the combined results from the extractions in your prac class and give a mean DNA concentration for each tissue type.
- Text describing your results. Make comparisons between the different tissue types, which tissue resulted in the greatest yield of DNA, and did the quality of DNA vary among tissue types? You will also need to refer to your table and figures when describing your results in the text.
Section 2
- A copy of your gel clearly labelled showing the PCR product (for the three tissue types), positive controls (male and female), negative control and size marker.
- Determine the sex for each of the samples shown on the gel.
- Text describing your results. What is the approximate base pair size of the W and Z genes, how many samples were male/female. Were there any differences in amplification between tissue types? Did all bands amplify? Did any of the negative controls amplify?
- Do the results obtained from the PCR correlate with the actual sex of the individual? Refer to your table and figures when describing your results.
Discussion section - In the discussion you need to discuss your results and compare them to other studies. Again, use sections to break up the discussion into logical issues and deal with them one by one. Include in your discussion:
- Was there any difference in the concentration of DNA extracted from the three tissues types? If so which tissue had the highest DNA concentration, which had the lowest? Was the observed result consistent with expectations?
- If no band was visible on the gel for a particular tissue type does this result mean that the extraction was unsuccessful, explain.
- Why is it important to store samples intended for genetic analysis correctly? Discuss how incorrect storage of samples can result in DNA degradation? Was DNA degradation evident in any of the tissue types used in this experiment?
- Why would DNA extracted from the blood spot on a feather provide DNA of higher quality than that extracted from the tip?
- Briefly discuss (using dot points) the pros and cons of using different tissue types for genetic studies of wild animals (think destructive, non- destructive (invasive) and non-invasive sampling).
- Explain (using dot points, short sentences) how the universal primers used in this experiment are able to provide information about the sex of an individual.
- Discuss (using dot points, short sentences) the advantages of employing universal sexing molecular markers.
- Was there evidence of genotyping errors in the sexing results from Gallus gallus? What type of samples are prone to these types of genotyping errors, why? What precautions should be taken to minimize errors?
- Include at least three references, with at least two of them being different from those in the introduction.
References - Reference section can be smaller font size. You will need a minimum of 5 Journal references (not including the practical manual). Look at the guidelines for referencing formats at the end of the manual.
Visual presentation - Please make sure to choose a colour scheme that is visually appealing (e.g. green and pink might not match that well), and create a poster that is easy to follow. Refrain from overcrowding the slide with large blocks of gray text, make sure your images are large enough and easy to understand. Word count should be about 800 - 1000 words, text is clear and to the point. Use of bullets, numbering, and headlines to make it easy to read. Use graphics colour and fonts effectively to achieve a consistent and clean layout.
Attachment:- Genetics and Genomics Assignment Files.rar