Please clarification those for me. I. A1 AND A2-TYPE OF Β-CASEIN EXPLAINED [Include some background information on β-casein and the difference (including at the amino acid level) betw

Prac: Gene -assisted selection for a2 milk 1 Gene -Assisted Selection for a2 Milk ™ Within this booklet you will find: • Instructions on pre -prac activities • Lab safety information • Pipetting manual • Laboratory manual – please keep in mind that this manual is desig ned for face to face classes taking place in the lab. Although we are not able to carry out lab activities in person this semester, it is important that you read through the manual to get a b etter understating of the steps involved and how each of the lab techniques is performed. This will also help you understand the work -flow in the prac videos on LMS. • Post -prac activities and assignment guidelines Wikimedia Commons 2 Prac: Gene -assisted selection for a2 milk 1. What is this prac class about? Once you have worked through the online prac videos and the questions within this booklet, you will need to write a diagnostic report for the farmer based on your findings; the diagnostic report is an individual assessment and will make up 7% of your overall mark. The prac videos and manual will go through the following te chniques and concepts: • Basic pipetting – a technique frequently used in the lab • Lys is – (= break open) sperm cells from bulls to release the DNA; • Setting up a PCR (polymerase chain reaction) for the amplif ication of β -casein alleles. • Loading samples on an agarose gel; • Analysing PCR products via gel electrophoresis; • Visualising and interpreting the DNA bands on the gel images 2. Pre -prac activities There are two pre -prac activities you need to complete before you begin to work through the A2 milk prac conte nt: (1) watch the lab safety induction (‘Good Laboratory Practice’) on the LMS, and (2) read the information in this prac manual, including supplementary online resources (see further for more details). To prove that you have done both, you will need to: 1) Complete the lab safety quiz on the LMS. You will need to obtain a score of no less than 100% before you get access to the pre -lab quiz. You can try the lab safety quiz as many times needed. 2) Complete the A2 milk pre -prac quiz on the LMS. You will have 30 min to complete the quiz which is due by Friday 19 th March 11:55pm. The quiz will go towards 3% of your overall mark. Wikimedia Commons Prac: Gene -assisted selection for a2 milk 3 2.1. Lab safety induction As a researcher it is important to familiarise yourself with how to conduct experiments safely in a laboratory set ting. Watch the lab safety induction on the LMS (a Powerpoint presentation named ‘Good Laboratory Practice’). You will learn about wearing the appropriate personal protective equipment (PPE) in the lab as well as what you should and shouldn’t do when worki ng in a laboratory environment. 2.2. Background knowledge Before beginning the work associated with the A2 milk prac, y ou need to have sufficient background knowledge on (a) a2 Milk™, (b) gene -assisted selection, and (c) the experimental procedures. (a) Bac kground on a2 Milk™ a2 Milk™ is natural dairy milk produced by the New Zealand a2 Milk Company™. You will probably have seen this brand of milk in supermarkets or on TV. Check out the following websites and try to find answer s to the questions below. www.a2milk.com.au (a2 milk company) https://a2milk.com.au/health -professionals/ (a2 milk company) http://www.legendairy.com.au/ dairy -foods/dairy -products/milk/a1 -and -a2 -milk -proteins (Dai ry Australia) • What are the different categories of milk proteins? • What does the 'a2' in a2 Milk™ refer to? How is a2 Milk™ different to 'regular' milk? • What causes the variation in this type of milk protein? • How is a2 Milk™ made? • What breeds of dairy cattle have the highest amounts of the A2 -type protein in their milk? • What are the alleged consumer's benefits from drinking a2 Milk™ instead of 'regular' milk? • Is a2 Milk™ a suitable alternative for people who are lactose -intolerant? (b) Gene -assisted selection The analysis you will conduct during these lab sessions is an example of genotyping animals in order to set up a gene -assisted selection program. Revise the definition of genes , alleles and genotypes as well as the section on marker - and gene -assisted selection in Chapter 2: Fundamentals of Animal Breeding (refer to the theory notes). (c) Experimental procedures It is important to have a general understanding of what the practical classes are about and what the intention and principle is of the different steps of the experiment before you attend the lab sessions or in the case of Tip: Write down the answers to these ques tions on a separate piece of paper and keep it in a binder together with this prac manual! 4 Prac: Gene -assisted selection for a2 milk online pracs before you start to comp lete your quizzes and assignment . This way, you will work more efficiently and will have a better overall un derstanding of the key concepts covered in this prac. The prac will cover a number of commonly used molecular biology techniques and terminology, so make sure you are well prepared! Carefully read through this practical manual and watch the videos referred to in the manual. GUIDE FOR THE MASTER PIPETTER 10 Prac: Gene -assisted selection for a2 milk For the techniques that you will be performing in the lab pracs, you will frequently need to pipette very small volumes using adjustable micropipettes . These pipettes are indispensable tools in biotechnology research. A range of models and volumes are available. In molecular research, it is very important to pipette solutions accurately and to prevent the contamination of solutions. Therefore, we will begin this prac with some exercises in pipetting technique and conversion of metric volumes. CHECK IT OUT Have a look at the characteristics of one of the micropipettes on your bench and look for the different parts ( Fig. 1). Wear gloves when handling the pipettes. Do not turn the plunger yet! Figure 1. Features of a micropipette. The top of the pipette (or the side of the pipette, depending on the brand) displays the volume range and the set volume can be read from the display. Always verify that you are using the correct and most accurate micropipette for the volume that you require. There are different types of pipettes with different volume rang es available in the prac lab. For example: Prac: Gene -assisted selection for a2 milk 11 2 – 20 µl with increments of 0.02 µl Maximum volume will be displayed as: 20 – 200 µl with increments of 0.2 µl Maximum volume will be displayed as: Decimal point 100 – 1000 µl with increments of 2 µl Maximum volume will be displayed as: Since different brands of pipettes are used in the prac lab it is important to ALWAYS verify the volume range as well as the display of the pipette, which may or may not show a decimal point. 1. Volume conver sions In molecular biology protocols, the most common units of measurement are millilitre (ml) and microlitre (µl). As you will need to correctly pipette the volumes indicated in the prac manual, it is important to familiarise yourself with the conversion of the metric units of measurement. 1 litre = 1,000 ml = 1,000,000 µl EXERCISE Complete the following exercises and show your results to a demonstrator before proceeding to the next step! EXERCISES 1. Convert the following volumes. 1 ml = litre (l) 2.5 ml = µl 14 µl = litre (l) 911 µl = ml 2. If you need to pipette 0.5 ml of a solution using a 100 – 1000 µl pipette as shown on the right, what should be shown on the display? 12 Prac: Gene -assisted selection for a2 milk 3. What volume does the following reading of a 2 – 20 µl pipette represen t? 4. Which of the following pipettes will you use when taking up 24 µl of a solution? 2. Pipetting technique VIDEO Check out this video by the University of Leicester about good micropipetting technique: Using a Micropipette (http://www.you tube.com/watch?v=uEy_NGDfo_8 ). When using a micropipette to dispense liquid keep the following in mind: • Before using a micropipette, always apply a tip first! Never dip the pipette itself in a fluid! • Always ensure that you set the volume ONLY within the range that is indicated on the side of the pipette. Turning the knob outside this range will damage the pipette. These pipettes are very expensive!! • Always keep the micropipette vertically when there is fluid in the tip; otherwise the liquid can accidentl y run back inside the pipette, damage and contaminate it. Do not lay a liquid -loaded pipette down on the lab bench. • Let the plunger rise SLOWLY after taking up fluid with the pipette. Do not let it snap back; otherwise fluid can get into the pipette result ing in damage and contamination. • ALWAYS take a new tip each time you have pipetted a solution. If you use the same tip, you will contaminate a solution with the previous liquid you have pipetted! Prac: Gene -assisted selection for a2 milk 13 2.1. Step -by -step instructions for pipetting a solution Set the volume of the pipette and attach a pipette tip. Hold the micropipette in one hand with the volume correctly set. Push the end of the pipette firmly into a disposable tip. The types of tips available in the prac lab are described in section 2.2 below. Press the plunger of the pipette to the first stop. Depress the plunger to the first stop as indicated in Figures 2 (B) and 3. You will feel some resistance when reaching the 1 st stop. Keep the plunger depressed and holding the pipette vertically immerse the tip only a few mm into the sample liquid. Do not submerge the tip fully in the solution; the tip should not touch the bottom of the bottle or tube. Open up the tube/bottle you are pipetting liquid from as well as the tube you are expelling solution in in advance so that you don’t have to open lids with your pipette in one hand! Figure 2. Pipetting technique. (A) Plunger released. (B) Plunger depressed until first stop. (C) Plunger depressed until second stop. Figure 3. Pipetting technique for taking up and expelling liquids. 14 Prac: Gene -assisted selection for a2 milk Release the plunger slowly to take up the liquid. Allow the plunger to return SLOWLY to the up position ( Fig. 3 ② ). Be careful to not let the plunger snap back as this will cause liquid to enter the pipette! Always lo ok at the liquid in the pipette tip to see if you have pipetted the volume correctly and that there isn’t a large air bubble present because the tip wasn’t fully submerged in the liquid. This will cause you to pipette an inaccurate volume. To avoid errors when pipetting, hold the solution you are pipetting in one hand and observe the solution being taken up by the pipette tip. Expel the liquid by pushing the plunger to the first and then the second stop. 1) While holding the pipette vertically, touch the mi cropipette tip to the inside wall of the receiving vessel into which you are going to expel the sample ( Fig. 3 ③ ). 2) SLOWLY depress the plunger to the first stop. ( Fig. 2 B – 3 ④ ) 3) Wait one second and then press the plunger to the second stop ( Fig. 2 C – 3 ⑤ ), expelling any residual liquid. With the pipette fully depressed , withdraw the tip from the tube and allow the plunger to return to the up position. It is important to NOT let the plunger return to the up position when the 2 nd stop is reached and the pipette tip is still in the solution. The liquid will be taken up again and might not be able to be expelled anymore! Discard the pipette tip. Discard the pipette tip in the appropriate container by pressing the ejector button. Use a fresh tip each time to avoid contamination! 2.2. Types of pipette tips in the prac lab There are several pipette tip sizes available depending on the micropipette you are using. The most commonly used pipette tips in the prac lab are: Yellow tips: use with the 2 – 20 µl and 20 – 200 µl pipettes Blue tips: use with the 100 – 1000 µl pipette Autoclaved/sterilised pipette tips The pipette tips used in this practical class to analyse the bull semen samples have been autoclaved (sterilised), a process that also destroys deoxyribo nucleases (DNases), enzymes that degrade DNA. A common source of DNases is human contact, for example through saliva, hair, skin and nails. Degradation of DNA can lead to low yields in the techniques that you will be performing in this prac, so it is cruci al to kee p the pipette tips sterile, and to make sure you work in a clean way (not touching your hair with your gloves, for example). Prac: Gene -assisted selection for a2 milk 15 Make sure that the pipette tip does NOT come in contact with anything apart from the solution you are going to pipette. If it does (for example when the pipette tip touches your gloves, the bench, your lab coat etc .), you can contaminate your solutions with bacteria, DNases and other contaminants. Do not touch the tip once it is on the pipette, again to avoid contamination. Close the pipette tip box when you are not using it. When working in a molecular lab it is crucial that you do not contaminate solutions and your samples, as this will greatly impact the validity of your results . PRACTICAL EXERCISE Now practise your mi cropipetting technique by following the protocol below. Once you have completed this exercise and had your results checked by a demonstrator, move on to the main manual for the next exercise. PROTOCOL MATERIALS: • Micropipettes • Yellow and blue pipette tips in containers • Microcentrifuge tubes • Waste containers • Small beakers • Distilled water 1. Take a beaker and dispense some distilled water. 2. Practice pipetting different volumes of water into empty microcentrifuge tubes. Make sure that there are no air bubbles in the pipette tip when you take up your solution! Ask a demonstrator to verify your pipetting technique. 3. Pipette the volumes indicated below in separate microcentrifuge tubes. Write the volume dispensed on the lid of the tube. Ask a demonstrator to check the tubes once you have finished. Note that each student needs to prepare a set of tubes. 2 µl 20 µl 417 µl 950 µl 4. Once you have completed the exercise, empty the beakers with water in the sink and return the beakers to your workstation. LAB ORATORY MANUAL Prac: Gene -assisted selection for a2 milk 17 The protocols described below are based on the methods used by Lien et al. (1992) for detecting multiple β-casein alleles. 1. Lysis of bull semen The first step in determining whether your bull has A1 , A2 or both alleles of the β -casein gene , involves releasing the genomic DNA (the DNA contained in the nucleus of the cell, which contains the β-casein gene) from the sperm cells. So, we start by breaking open or ‘lysing’ the sperm cells ( Fig. 1) . PRACTICAL EXERCISE For this practical sessi on you will work in pairs. Each pair of students will take one semen straw for analysis. Follow the protocol below to lyse the sperm cells. MATERIALS: • Micropipettes • Sterile yellow and blue pipette tips in boxes • Semen straw • Empty tubes • Wash solution • Lysis solution • Scissors • 70% ethanol • Microscope • Waste containers • Biohazard bag for disposal of biological materials 1. Each team will receive 1 bovine semen straw ( Fig. 2) with the name of the donor bull. These straws have been stored at -80°C for preservation and need to be thawed first. The straw contains ± 200 µl of semen. Each team should take from the workstation: • 1 semen straw; • 1 pair of scissors; • 1 x empty 1.5-ml tubes. 2. Use one of the permanent markers, which you can find at the workstation, to write your team number on the lid of the empty tubes. Write legible! 3. Open the empty 1.5 -ml tube. 4. Cut open the semen straw right below the plug(s), as shown: Figure 2. Semen straws. Figure 1. Bull semen PROTOCOL 18 Prac: Gene -assisted selection for a2 milk 5. Place the straw with the side that has been cut open into the test tube. 6. Now cut open the other end of the straw. The semen sample will pour out of the straw. Wipe the scissors with 70% ethanol and dispose of the straw in the biohazard waste bag. If you would like to have a look at the sperm cells in your bull sample: Take 5 µl from the semen sample and pipette in a separate 1.5 -ml tube. Place this tube in the ice container at your workstation. You can then use th is aliquot later on to look at the semen cells under the microscope with the help of a demonstrator. 8. Wash the sperm cells by adding 750 µl of wash solution to the sample. You can find the wash solution at your workstation. Wash solution = 2% sodium dodecyl sulphate (SDS) in phosphate -buffered saline (PBS) SDS is a denaturing detergent that is highly flammable, harmful in contact with skin and if swallowed, and irritating to eyes, respiratory system and skin. 9. Place the tube with the sperm in wash solution on the rocking platform for 10 minutes (use some masking tape to stick it to the platform). 10. Bring your sample to the centrifuge and the demonstrator will set up the machine. Centrifuge the tube for 10 minutes at 3,000 g.* 11. You will notice that a small white pellet is visible at the bottom of the tube after centrifuging. 12. Remove the supernatant by slowly taking up the solution above the pellet ( Fig. 3). Dispense the supernatant in the waste container. Be careful not to touch the pellet with the pipette tip! 13. Add 1 ml of lysis solution to the pellet and vortex the sample until the pellet is fully resuspended. You can find the lysis solution at your workstation. Lysis solution = 1% Tween + 15 mM DL -dithiothreito l (DTT) + 500µg/ml proteinase K in phosphate -buffered saline (PBS) DTT is harmful if swallowed, and irritating to eyes, respiratory system and skin. 14. Incubate the sample for 1 hour at 60°C in the water bath (in a floating rack). Invert the tube 5 time s every 10 minutes. While waiting, complete the exercises on pages 18 and 20 -23. During this time your demonstrator will help you with visualising the sperm cells under the microscope. 15. Place the samples in a boiling water bath (in a floating rack) for 5 mi nutes. 16. Vortex the sample for 1 minute and proceed immediately with the PCR (p. 24). *Note: 3000xg refers to the relative centrifugal force measured in acceleration relative to gravity (g). This is the force exerted on the contents of the rotor, resulting from the revolutions of the rotor. Figure 3. Removal of supernatant Prac: Gene -assisted selection for a2 milk 19 EXERCISE Complete the following exercises on the methodology of cell lysis and show your answers to a demonstrator before leaving the lab! EXERCISES 1. What is the role of Tween (a detergent) and proteinase K (a protei n-digesting enzyme) in the lysis solution? [Tip: What do you use a detergent for?] 2. Why are the samples incubated at 60°C after adding the lysis solution and boiled for 5 minutes after the incubation? 2. PCR Once the DNA is released from the sperm cells you will use a polymerase chain reaction (PCR) to make multiple copies of a relevant segment of the β -casein gene. The DNA segment that you will amplify is 251 base pairs long (1) and the full β -casein gene is about 32 times the size of that fragment. Think of it as follows: Using PCR, a specific fragme nt of the β -casein gene can be amplified exponentially so that more and more (millions) copies of that fragment are produced. Amplifying a fragment will allow us to produce enough copies of the fragment to visualise this via gel electrophoresis (practical session 2). VIDEO The following resources will provide you with some more information about the principle of PCR . Go through these resources before coming to the lab session. • PCR animation (http://highered.mcgraw - hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.h tml) • Another PCR animation (http://www.youtube.com/watch?v=2KoLnIwoZKU) • Setting up a PCR reaction (http://learn.genetics.utah. edu/content/labs/pcr/) 1 The length of DNA fragments is expressed in base pairs (bp). 20 Prac: Gene -assisted selection for a2 milk 2.1. The principle of PCR In the animated resources above you will have seen that a PCR reaction consists of several cycles that each include the same three steps: 1) Denaturation, 2) Annealing and 3) Elongation. DENATURATION DNA is double -stranded (think of it as a zipper or a ladder) and during the first step in the PCR reaction the temperature is increased to 94°C which makes both strands of DNA separate ( Fig. 4, ) by breaking the hydrogen bonds that hold the strands together. This is called ‘denaturation’. ANNEALING After the initial denaturation step, the temperature is decreased to 62°C for annealing of the two primers. During the annealing step, smal l pieces of DNA, called primers , will bind or ‘anneal’ to the (complementary) ends of the t arget DNA sequence and will form the starting blocks to make new DNA strands ( Fig. 4, ). ELONGATION During the elongation step the temperature is increased again to 72°C, which is the optimal temperature for the enzyme Taq DNA polymerase to work. The enzyme adds additional nucleotides (the building blocks of the DNA) to the end of each primer in order to generate a segment of double -stranded DNA ( Fig. 4, ). Durin g the PCR reaction, steps 1 to 3 are repeated for 40 cycles. Prac: Gene -assisted selection for a2 milk 21 Figure 4. The principle of the polymerase chain reaction (PCR). EXERCISE Complete the following exercises on the principles of PCR and gene -assisted selection . Show your results to a demonstrator before leaving the lab! EXERCISES 3. Remember the animations of the PCR principle? List the different components that need to be added to a PCR reaction to allow amplification to occur. • • • • • 4. If you wanted to investigate whether yo ur bull carries the gene for the genetic disease arachnodactyly using PCR, what would you change in the PCR reaction? 22 Prac: Gene -assisted selection for a2 milk 5. Make a schematic drawing of a DNA double strand and the position of primers for amplification of a segment of this DNA. You would now like to amplify a shorter piece of this same segment of DNA. Where would you position your primers? Indicate this in your drawing. 6. Why do you need two primers in your PCR reaction and not just one? 7. For the PCR reaction that you will be runni ng in this lab session, you will use a PCR program that exists of 40 cycles of denaturation - annealing -elongation. To obtain more DNA, do you think you can let the PCR run for a much longer period of time? Why or why not? [Tip: Have a look at the image on the right] 8. Figure 4 (next page) shows the amplification of a target sequence by PCR. • Indicate in Figure 4: o The cycle number; o Which DNA double strands, produced during the various cycles, are actually of the correct target size , i.e. the size o f the fragment that you want to amplify? • Use the information in Figures 3 and 4 to draw a graph ( x-axis : number of cycles; y- axis : number of double strands of DNA) in which you indicate the total number of double strands for each cycle with a dot ( ) and the number of double strands of the correct target size with ‘ ’. Then connect the circles in the graph. Prac: Gene -assisted selection for a2 milk 23 Figure 4. Amplification of a DNA fragment in a PCR reaction. 24 Prac: Gene -assisted selection for a2 milk Draw your graph here: 2.2. PCR on lysed sperm cells After completing the final step of the lysis reaction on page 18, you can start setting up your PCR reaction. For your PCR reaction, you will use two primers (that act as starting points for the enz yme to make the new DNA strand) designed specifically to bind to a section of the β -casein gene in which there are differences betwe en the A1 and A2 alleles. These primers are the same ones as used by Lien et al. (1992) (and called CASB67 and CASB122 in this paper). The temperature at which these primers will bind to the DNA is 62°C (= annealing temperature). PRACTICAL EXERCISE Use the lysate that you have prepared to set up the PCR reaction following the protocol below. Prac: Gene -assisted selection for a2 milk 25 PROTOCOL MATERIALS: • Micropipettes • Sterile yellow and blue pipette tips in boxes (autoclaved) • Container for ice • PCR tube • Sterile ultrapure water (H2O) • PCR Master Mix (M) • Small rack for PCR tubes • Markers 1. While one of your team members is vortexing the sample (p. 18), take the following items (per team) from your workstation: • 1 plastic container that you fill with ice; • 1 rack for PCR tubes; • 1 empty PCR tube (200 -µl tube). 3. Use one of the permanent markers at your workstation to write your team number on the lid of the PCR tube. For example, team 2 on the Wednesday prac session: W2. Write legible! 4. Carefully pipette in the PCR tube: • 35.3 µl of ultrapure water from the tube labelled ‘H2O’ • 12.7 µl of Master Mix from the tube labelled ‘M’ • 2 µl of lysed sperm cells (just vortexed!) The Master Mix contains 1x PCR buffer, 0.2 mM nucleotide mix, 1.5 mM MgCl 2, 0.5 µM primer CASB67, 0.5 µM primer CASB122 and 1 unit Taq DNA polymerase. Place this tube on ice immediately and keep it on ice until it will be placed in the thermal cycler. This tube contains the necessa ry PCR components including the Taq DNA polymerase. 5. Mix the reaction mixture by slowly pipetting up and down. 6. Bring your tube to the demonstrator who will set up the centrifuge. Centrifuge the tube briefly to make sure that all the components are at the bo ttom of the tube. Place the PCR tube in the thermal cycler. The demonstrator will set up the thermal cycler with the following PCR program: Number of cycles Temperature Duration PCR step 1x 94 C 3 min Initial denaturation 94 C 45 sec Denaturation 40x 62 C 30 sec Annealing 72 C 1.5 min Elongation 1x 72 C 10 min Final elongation 1x 4 C (Storage) 26 Prac: Gene -assisted selection for a2 milk 3. Restriction digestion Next, the PCR products that have been generated in the previous prac session are cut with the restriction enzyme Taq I. Restriction enzymes , also known as restriction endonucleases , are DNA - cutting enzymes found in and harvested from bacteria. A restriction enzyme recognizes and cuts DNA only at a particular DNA sequence. For example, the commonly used restriction enzyme Eco RI cuts a DNA sequence as follows: The Taq I restriction enzyme used in this experiment, on the other hand, cuts its recognition sequence like this: VIDEO The following resources will provide you with some more information about the concept of restriction digestion . • Animation of restriction digestion ( http://highered.mcgraw - hill.com/olcweb/cgi/pluginpop.cg i?it=swf::640::480::/sites/dl/free/0073383074/811328/restri ction_endonucleases.swf::Restriction%20Endonucleases) (first few seconds only) • Animation and exercise on restriction digestion (https://stwww1.weizmann.ac.il/geneengine/wp - content/uplo ads/sites/27/articulate_uploads/Restriction_EN5/story.html ) The DNA fragment amplified by PCR in the previous lab session has a length of 251 base pairs (bp), regardless of whether a fragment of the A1 or A2 allele of the β-casein gene was amplified . Th e PCR reaction carried out by everyone in the class therefore generated the same 251 -bp fragment. Then how can we find out whether the bulls have A1 , A2 or both alleles of the of the β-casein gene? Performing a restriction digestion on the PCR product with the Taq I restriction enzyme allows us to discriminate between the A1 and A2 allele of β-casein. A Taq I restriction site is present in the amplified A1 sequence, but not in the A2 sequence (2). Treating the PCR products with Taq I will therefore generate two DNA fragments if the A1 allele was amplified, but will have no effect when the A2 allele was amplified: 2 The Taq I restriction site is not naturally present in the A1 sequence. We have artificially introduced it by using PCR primers designed for this pu rpose. See Lien et al. (1992) for more details. Prac: Gene -assisted selection for a2 milk 27 The following procedure will be used for the Taq I restriction digestion. This procedure will be carried out by the demonstrators before the start of the second lab session, due to time restrictions. PROT OCOL (Carried out by demonstrator) 1. In a sterile tube, combine the following components: • 15.7 l of sterile, ultrapure water • 3 l of 10x restriction enzyme buffer • 0.3 l of acetylated bovine serum albumin (10 g/ l) • 10 l of PCR product The restriction buf fer contains 6 mM Tris -HCl, 6 mM MgCl 2 and 100 mM NaCl (pH 7.5). Bovine serum albumin (BSA) is added to some restriction reactions to prevent adhesion of the enzyme to reaction tubes and pipette surfaces. BSA also stabilises some proteins during incubation . 2. Mix the reaction mixture by slowly pipetting up and down and then add 1 l Taq I restriction enzyme (10 U/ l). 3. Mix gently by pipetting, close the tube and centrifuge for a few seconds in a microcentrifuge. Incubate for 5 hours at 65°C. 4. Gel electrophoresis 4.1. Principle After digestion with Taq I restriction enzyme, the DNA fragme nts will be separated via gel electrophoresis and visualised so that their size can be determined. Based on the fragment size observed after gel electrophoresis you can determine whether your bull has the A1A1 , A1A2 or A2A2 genotype. 28 Prac: Gene -assisted selection for a2 milk During gel electroph oresis, DNA fragments are forced through a matrix of agarose (sugar chains) by an electric current. The buffer in the electrophoresis chamber conducts the electric current (by providing ions), reduces pH changes and prevents overheating of the gel by the e lectric current. Smaller fragments will migrate through the matrix more easily than larger fragments since these experience more resistance when passing through the agarose. The percentage of agarose in a gel will influence the resolution, i.e . the separat ion of small or large fragments of DNA on the gel. The agarose matrix acts as a sieve so the greater the agarose concentration the smaller the pores that are created in the matrix and the higher the resistance the DNA molecules experience (and vice versa). The percentage of agarose in the gel therefore affects the separation of small and large DNA fragments differently ( Fig. 5) . When samples are loaded on a gel, a loading dye is added to the sample. This dye usually contains Ficoll (a polysaccharide) or g lycerol to make the sample ‘heavier’ so that it sinks into the well when the sample is loaded. The solution also contains bromophenol blue, which is a standard tracking dye for electrophoresis. The loading dye allows the researcher to easily see the sample s when loading them on the gel ( Fig. 6a) and to keep track of how far the DNA has migrated through the gel. Another dye is added to the agarose gel in order to visualise the DNA bands after gel electrophoresis ( Fig. 6b) . We will be using the SYBR®Safe DNA gel stain, which can be visua lised using blue light or UV excitation and is also safer to use than the traditional dye ethidium bromide. Figure 6. (a) Loading of samples containing a blue loading dye on an agarose gel. (b & c) Migration of DNA samples through the agarose gel matr ix (note that migration can be tracked using the loading dye (blue colour)). (d) Placing the agarose gel in the gel imager for visualisation of DNA fragments. (e & f) DNA fragments visualised with UV illumination after gel electrophoresis due to the incorporat ion of a DNA gel stain (Wikimedia Commons). Figure 5. Example com parison of the separation of DNA fragments (100 bp to 600 bp) on a 0.7% versus a 1.5% agarose gel. Prac: Gene -assisted selection for a2 milk 29 Besides the DNA samples, a DNA ladder will be included on the gel. A DNA ladder is a solution that consists of various DNA fragments of known size, which is run alongside samples so that the size of the DNA fra gments in the samples can be estimated. The DNA ladder that we will be using is a 50 -bp or 100 -bp DNA ladder ( Fig. 7) . 4.2. Pouring an agarose gel In this practical session, we will be using an agarose gel that has been prepared in advance. However, the demonstrator will show you how the agarose gel is made. Take notes below during the demonstration. There might be questions about this in the post -lab quiz! VIDEO This video shows you some additional information about preparing an agarose gel: Making an agarose gel (http://www.youtube.com/watch?v =wXiiTW3pflM) . Figure 7. 50 -bp and 100 -bp DNA ladder (Bioline). 30 Prac: Gene -assisted selection for a2 milk Figure 8. Gel electrophoresis tank. 4.3. Loading samples Before preparing your sample for gel electrophoresis, you will practice loading samples in the wells of a pre -made agarose ge l. Make sure to practise enough! One person of your team (it might be you!) will have to load the actual sample on the class agarose gel. PRACTICAL EXERCISE Practice loading samples into wells of an agarose gel following the protocol below. PROTOCOL MATERIALS: • Micropipettes • Yellow pipette tips in containers • Practice loading dye – Tube labelled ‘LD -P’ • Tube rack • Gel electrophoresis tank with agarose gel 1. Each team should go to a central workstation where a gel electrophoresis tank has been set up with a practice gel. 2. Pipette 20 l of practice loading dye in one of the bottom wells of the agarose gel. Use the pipette tips in the containers, not the sterile pipette tips! Ask your demonstrator for help with this. Prac: Gene -assisted selection for a2 milk 31 Make sure no air bubble is present in the end of the pipette tip. If there is, just push the air out slowly by pressing the plunger and keep the plunger pressed in like this when you dispense the solution into the well. Load several wells with the practice loading dye solution. Take a new pipette tip each time ! When pipetting a sample in a well, do not place the pipette tip fully in the well as you could puncture the well and the bottom of the gel. This could lead to your sample draining into the buffer. It can also cause the sample to swirl up (because of the turbulence) and squirt out of the well. Keep the pipette tip just above or just inside the well and slowly dispense the sample. The glycerol in the loading dye makes the sample sink to the bottom of the well. Once you are finished practicing, you will need to start preparing your sample to load on the gel. PRACTICAL EXERCISE Prepare your sample for gel electrophoresis by following the protocol below. PROTOCOL MATERIALS: • Micropipettes • Sterile yellow pipette tips in box • Loading dye (5x concentrated) – Tube labelled ‘ LD ’ • Empty 1.5 -ml microcentrifuge tube • Tube rack • Restriction digest 1. Per team, take the following from the workstation: • 1 empty 1.5 -ml tube; • the PCR digest from the sample that your team has prepared during the previous prac session (the group number is indicated on the lid of the tube); 1 tube with loading dye, labelled ‘LD ’. 2. Write your group number on the lid of the empty tube. 3. Pipette 20 l of the digest into this tube. 4. Add 5 l of the loading dye and mix by pipetting up and down. The loading dye can be found at your workstation. Do NOT use the practice loading dye solution for this step. The loading dye (6x) has the following composition: 2.5% Ficoll® -400, 11 mM EDTA, 3.3 mM Tris -HCl, 0.017% SDS, 0.015% bromophenol blue (pH 8.0 at 25°C). This solution contains SDS, which often results in sharper bands, as some restriction enzymes are known to remain bound to DNA following cleavage. EDTA is also included to chelate magnesium (up to 10 mM) in enzymatic reactions, thereby stopping the reaction. 5. Bring your tube to the centrifuge for a quick spin before loading the sample on the gel. The demonstrator will set up the centrifuge for you. 6. Bring you r sample to your workstation for loading on the gel. 32 Prac: Gene -assisted selection for a2 milk 7. A demonstrator or a student will add 6 l of the 50 -bp or 100 -bp DNA ladder solution to the first and last well of the gel. The DNA ladder solutions also contain loading dye. 8. Dispense your sample in the appropriate well using a sterile pipette tip. Once all the samples have been loaded on the gel, the electrodes will be connected to the power source and the voltage will be set at 100 V. The gel will run for at least one hour. Complete the exercises on p. 32 - 34 during this time. You can track the movement of the DNA by keeping an eye on the migration of the loading dye. VIDEO This video shows you how to run an agarose gel: Running an agarose gel (http://www.youtube.com/watch?v=U2 -5ukpKg_Q) . 4.4. Imagi ng of the gel Once the gel electrophoresis has been completed, the DNA fragments can be visualised by UV illumination. We will use the Gel Doc EZ System (Bio -Rad) and the UV sample tray. As this is a ‘closed’ imaging system, no protective eye gear is neces sary when using the UV light. The image of the gel will be saved and uploaded on the LMS. After the gel has been removed from the sample tray, the tray is cleaned with 70% ethanol, dried and stored. EXERCISE Once the image has been made, compare the sizes of the generated DNA fragments with the DNA ladder and deduce which alleles of -casein are expressed in the various bulls. List the different bulls and their -casein genotypes on a separate piece of paper. EXERCISE Complete the following exercises and show your results to a demonstrator before leaving the prac class! EXERCISES 9. What is the size of the fragment(s) generated after restriction digestion when your bull has the A1A1 , A2A2 or A1A2 genotype? • A1A1 • A2A2 • A1A2 10. Indicate next to the 50 -bp and 100 -bp ladders in Figure 7 where the A1 and A2 fragments are expected to be after gel electrophoresis of the restriction digests.  Keep in mind that an electrophoresis unit is filled with a highly conductive sa lt solution and that it uses DC current at high voltages ! Both supply leads should be connected BEFORE turning on the power supply. After use, turn off the power supply, and then disconnect both leads. Also watch out for fluid leaks or spills as they can act as pathways for current and electrical shock! Prac: Gene -assisted selection for a2 milk 33 11. The electrophoresis tank has a positive (red) and ne gative (black) electrode ( Fig. 8) . How should you place the gel (i.e. the wells closest to the negative or positive electrode?) in the electrophoresis chamber for the DNA to migrate through the gel, given the negative charge of DNA? 12. Suzie, a novice scientist, has performed a r estriction digestion on genomic DNA from six hair root samples to determine the presence of a gene that confers disease resistance. Suzie has run these digest samples on an agarose gel (see image). Suzie is unsure about the results as the image shows mostl y a smear of DNA. She doubts that her results are conclusive. What advice would you give to Suzie? _ 13. Factor XI (FXI) is one of more than a dozen proteins involved in the early blood coagulation cascade. Bovines with FXI deficiency have a blee ding disorder and may have lower calving and survival rates and increased susceptibility to infectious diseases. It was discovered by Patel et al. (2007) that there is a 77 -bp insertion in the FXI gene of animals with the disease (insertion = the addition of 1 or more nucleotides in a DNA sequence so a 77 -bp insertion means an additional 77 bp when the animals have the disease. ). The gel image below shows the results of a PCR on genomic DNA extracted from white blood cells of six Holstein -Friesian bulls (la nes 1-6; lane 7 is a DNA ladder). • Determine the genotype and phenotype of each bull. You can use ‘ F’ for the normal factor and ‘f’ for the mutated (disease) factor, when writing down the genotype for the animal. (Note that – similar to the β-casein allel es – this is a case of codominance and hence both alleles of the gene pair are expressed. Animals that have an Ff genotype will have both a normal and a mutated FXI protein.) Bull 1: Bull 2: Bull 3: Bull 4: Bull 5: 34 Prac: Gene -assisted selection for a2 milk Bull 6: • Which bulls would you exclude from your breeding programs? Why? 14. Briefly explain the principle of gene -assisted selection and how this relates to this lab session. 15. Sometimes gene -assisted selection is more useful than ‘traditional’ selection based on phenotypic measures. How does this apply to our a2 Milk selection experiment? 16. Why is it important for selective breeding to have multiple alleles for a particular trait? Prac: Gene -assisted selection for a2 milk 35 Post -prac activities After completing the two lab sessi ons, you are to: (1) write a diagnostic report about your results, to be submitted by Friday March 26 th at 11:55pm ; and (2) complete a post -lab quiz , testing your knowledge of the prac procedures, outcomes and context, also by Friday March 26 th at 11:55pm . Writing a diagnostic report General information • The diagnostic report you will write for these practical sessions on a2 Milk™ should be around 800 -900 words long (up to 1000 words maximum) and will be based on the results provided to use in the prac resources online. The report consists of three parts: (1) The first part of the report should be a letter directed to Maggie which includes a written - out interpretation of the results of the test and addresses Maggie’s query she has raised in her e - mail. You can refer in your letter to figures, tables etc. that are located in the technical part of the diagnostic report, i.e. the genotyping report. (2) The genotyping report should include information about the type of test that you have used to determine whether the bulls had the A1A1, A2A2 or A1A2 genotype, an overview of the results, some general information on interpreting the results and some background information on gene - assisted selection for a2 Milk breeding programs. Where appropriate, scholarly references should be included. (3) The invoice should include the cost of the analysis and the total price including GST (goods and services tax), as well as any comments you might have. ** The word count includes both the letter and the genotyping report. Text in figures , tables as well as the reference list is not included in the word count. • Note that we are asking you to write a diagnostic report – not a lab report ! The typical format of a diagnostic report is very different to that of the lab reports you will have to write for other subjects and the lab report format described on the Achie ✓[email protected] site. It is therefore crucial that you carefully rea d the guidelines below and that you use the report template available on LMS. Have both documents open side by side. • The rep ort should be submitted via the appropriate Turnitin link on the LMS (see further). • The report needs to be submitted by Friday 26th March at 11:55 pm . The submission link closes at that time and late submissions are therefore not accepted unless you have r eceived special consideration or an extension (see guidelines in the subject learning guide)! • Even though you are to use the results given to you, this is an individual assignment. Copying from fellow students (or other sources) is plagiarism and will be d ealt with accordingly. • Your report will be marked on language (15%) , formatting and referencing (10%), and - of course - content (75%). In total, the report will contribute 7% to your final subject mark. 36 Prac: Gene -assisted selection for a2 milk Language Write your report using professional lan guage. For the Genotyping report component, the Achie ✓[email protected] site has helpful information on how to write in the field of science: ‘Scientific writing style’ (https://latrobe.libguides.com/writing/science -technology ) ‘Academic Style’ (https://latrobe.libguides.com/writing/structure ) • Make sure to check your report for grammar and spelling errors as well as errors in sentence construction! A text full of errors does not look professional and brings down the whole feel of the report, even when all the content is correct. • Do not use quotes in your report. It is the intention that you write the report in your own words and this does not look very professional in this type of repo rt. Formatting and referencing • As mentioned above, use the appropriate template for your report, which is available on LMS. • Make sure your report looks professional, as if it was made by a commercial company. You can use colours for subtitles or in your tables, etc. • Follow the guidelines of the ‘referencing tool’ on the Achie ✓[email protected] site (https://latrobe.libguides.com/harvard ) for referencing in the Harvard style , i.e. how to include in- text citations an d how to format references in a reference list. Make sure you include scholarly references . No in-text referencing is needed in the letter component of the diagnostic report but you will need to use in-text referencing in the genotyping report. When prepar ing your reference list, make sure to arrange this in alphabetical order by the first author’s surname (and year). • Follow the guidelines available on LMS for formatting figures and tables correctly. • The following components should be included in your repor t: (1) Letter addressed to Maggie (see template for structure) a. Name and student ID. b. The date the letter was prepared. c. The recipient’s correspondence details (see Maggie’s e-mail, which you can find on LMS). d. The main body of the text addressing Maggie’s queries . (2) Genotyping Report (= technical part of the diagnostic report, see template and information below) a. Details of the submitted samples, including date of the analysis b. Test method used c. Test results d. Interpreting genotype results e. A1 and A2 -type of β-casein expl ained f. Gene -assisted selection for a2 Milk breeding programs g. Glossary h. Reference list (3) Invoice Information to include in the letter to Maggie Start the letter off with a greeting, followed by the content of the letter in which you inform Maggie of the ou tcomes of your analysis and provide an answer to the question she asked in her e-mail. Make sure you include some introductory and concluding sentences and use paragraphs to make the reading more attractive. Finish with the signing of the letter. Keep in mind that the language should be appropriate for a client. Outcomes of the analysis Tip: Carefully study the marking rubric (on the LMS) for this report before you start writing – it clearly shows you what we will be looking for in your report when marking! Prac: Gene -assisted selection for a2 milk 37 When describing the outcomes of the analysis to Maggie, explain which bulls are suitable for an a2 Milk TM breeding program and why. How many animals were identified for each of the unsuitable genotypes? Also inform Maggie about any inconclusive samples and what will happen with these. Maggie’s query on potential health benefits of a2 Milk Note that you need to address Maggie’s query sufficiently using information from at leas t 2-3 scholarly sources ( a paragraph of minimum 150 -200 words to address this query). In this paragraph you will have to integrate information from these studies and compare their outcomes. Make sure you are sufficiently detailed in your explanation (Wha t did these studies investigate and how did the authors investigate this? What were the major outcomes and how do these studies compare?, etc.). Be critical when analysing the evidence; is there enough evidence to draw major conclusions? Information to include in the Genotyping Report a. Information about the samples analysed Include details about the type of samples submitted for analysis, date of analysis etc. in the appropriate box in the report template. b. Diagnostic test used Give a short description of the analysis method used (full sentences must be used, no dot points or inclusion of tables); it is not necessary to explain the purpose of each of the steps in the analysis but include key information to repeat the experiment. Keep this very succinct (no more than 200 words) and include only the most important information. Refer to (cite) the scholarly paper on which this method is based (available on the LMS). A diagnostic test report for a human patient is available on LMS and gives you an idea on how to write this particular section. Have a look at this example as similar laboratory techniques are used. Another good resource when writing this section is the paper by Lien et al. (1992), on which the methodology is based. You can find this paper on the LMS. c. Test results & interpretation Start this section of with a few introductory sentences. Include images of the gels with the class results and a table with an overview of the various bulls and their genotypes. Use the IDs of the bulls, not student team num bers (these have no meaning to your client!). Use the symbols A1 and A2 to write down your genotypes, not other letters ( Pp , Aa , etc.) that would only confuse your client. When listing genotypes, remember a bull genotype always consists of two alleles. For example, a bull may have genotype A2A2 , but ‘A2’ as a genotype is impossible. d. A1 and A2 β-casein explained For this section, provide some scientific background information about A1 and A2 β-casein and how these two variants differ (including at the amino acid level; you can include an image with appropriate reference but you need to explain it in words as well). You don’t need to provide information in this section on various scientific studies that have investigated benefits of a2 versus a1 milk; describ e this in your letter to your client as a response to her query. You can, however, mention something about how these two types of beta -casein might be metabolised differently. 38 Prac: Gene -assisted selection for a2 milk e. Interpreting genotype results Include a table similar to the example found in t he HornPoll TM DNA test flyer (see link below as well as the LMS) in which you describe the different genotypes A1A1 , A2A2 and A1A2 . Ensure you correctly interpret the genotype. This means that you need to keep in mind that the information you write in this table needs to apply to bulls as well. Start this section off with a few introductory sentences. https://genetics.zoetis.com/australia/_locale - as sets/documents/pfz2132hornpollcattlea4web.pdf f. Gene -assisted selection for a2 Milk™ breeding programs Include in this section an explanation of how genotyping cattle (bulls and cows) helps farmers to determine which animals to use in an a2 Milk breeding program. Also describe how gene -assisted selection can benefit farmers in comparison with phenotypic selection. Use the information in the HornPoll TM DNA test flyer as a guide (see link above and the LMS). g. Glossary Include a short glossary list with some key words used in the report. Four words have already been included in the report template that require a short description. Choose 4 more words that you think would be appropriate and useful to include for a client. An example glossary list can be found in the pet genetic analysis report (from Orivet, Canine DNA testing & feline DNA testing) which has been uploaded on LMS. h. References Include a reference list with a minimum of 4 scholarly references, correctly formatted. Information to include in the invo ice Include the number of samples analysed, the analysis cost and the total price including GST (10%). Include any comments in the appropriate comment box, e.g. samples that need to be re-analysed. Turnitin and plagiarism All assignments in AGR1AAS need to be submitted electronically via Turnitin; you do not need to submit a hard copy. We will mark and provide feedback on your assignment via Turnitin as well. You can view your mark and feedback by going to the Turnitin submission link of that particular assignment and opening your submitted file. Make sure to go through your feedback so that you can use this to improve your next assignment! Turnitin software investigates similarities between your report and other sources. An originality report will be ge nerated when you upload your assignment and this will show you the % similarity of your writing with other documents in the Turnitin databases (textbooks, online resources, papers, or other student submissions). Note that there is no ‘cut -off percentage’ below which your report is ‘fine’. If you keep in mind to always reference AND paraphrase , you should have no issues. Make sure you understand how to paraphrase correctly by going through the information on these pages: http://latrobe.libguides.com/academic -integrity Very useful guide! http://www.latrobe.edu.au/students/admin/academic -integrity/referencing -help/paraphrasing You can submit files in the following format to Turnitin: Tip: Look at the example of a real diagnostic report on the LMS to get a better idea of how a diagnostic report is different from a traditional lab report! Prac: Gene -assisted selection for a2 milk 39 • MS Word (.doc and .docx) • WordPerfect (.wpd) • Portable Document Format (text based) (.pdf) • HTML (.htm) • Rich Text (.rtf) and Plain Text (.txt) Note that Pages files (Mac) are NOT supported and that we won’t be able to open up your assignment for marking! If you have issues with lay -out problems in Turnitin after submitting your file, convert your file to a PDF. All files submitted to Turnitin must be text -based. For more information, go to this page: www.latrobe.edu.au/students/it/teaching/turnitin It is crucial that the work you submit is your own work , which means you have to include t he appropriate references (in -text AND in a reference list) as well as paraphrase appropriately. Copying sentences from other sources and then editing a few words is not paraphrasing (and will be picked up by Turnitin)! You need to integrate information from different resources and write this in your own way/words (but in professional language). Post -prac quiz After completing the online A2 content, we will quiz your understanding of the experiments, their outcomes and the exercises in the prac manual. Plagiarism is a serious offence as it goes against scientific integrity. When plagiarism is detected, this will in all instances be reported to the departmental Academic Integrity Advisor, who will take appropriate action. Information about academic integrity and paraphrasing can be found here: http://latrobe.libguides .com/academic -integrity https://www.latrobe.edu.au/students/admin/academic -integrity https://www.latrobe.edu.au/students/admin/academic -integrity/referencing -help/paraphrasing