Lab 8 - Heredity and Genetics of Blood type and the rapid antigen test

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity Principles of Heredity and the Genetics of Blood There are several terms that need to be clarified so that you can more easily follow the exercise. A gene is a segment of DNA that directs the expression of a particular characteristic ( trait ). Genes are located on chromosomes, and the location where a particular gene is found is referred to as the locus (plural: loci) of that gene. An allele is a gene for which there is an alternative expression. For example, a diploid organism carries the allele “ A ” on one homologous chromosome, and the allele “ A ” on the other. The genotype of this organism is then AA and it is said to be homozygous . An organism may also carry two different alleles. For example, on one chromosome it could carry the allele “ A ” and on the other it could carry the allele “ a ”. The genotype of such an organism is then Aa , and it is described as heterozygous for this chromosomal locus. The genotype of an organism is the listing of the two alleles for each trait that it possesses. The phenotype of an organism is a description of the way a trait is displayed in the structure, behavior, or physiology of the organism. Some alleles are dominant to others and mask the presence of other alleles. The dominant condition is indicated by uppercase letters (e.g., “ A ”). The alleles that are masked are called recessive alleles. The recessive condition is indicated by lowercase letters (e.g., “ a ”). When both dominants are present in the genotype ( AA ), the organism is said to be homozygous dominant for the trait, and the organisms will show the dominant phenotype (trait expression A). When both recessives are present in the genotype ( aa ), the organism is said to be homozygous recessive for the trait, and the organisms will show the recessive phenotype (trait expression a). In the case of complete dominance, the dominant allele completely masks the recessive allele, and an organism with a heterozygous genotype ( Aa ) will show the dominant phenotype (trait expression A). The principles that govern heredity were discovered by a monk named Gregor Mendel in the 1860's. In today’s lab you will review the principles of heredity on a series of exercises. MONOHYBRID INHERITANCE Monohybrid inheritance is the inheritance of a single characteristic. The different forms of the characteristic are usually controlled by different alleles of the same gene. For example, a monohybrid cross between two pure- breeding plants (homozygous for their respective traits), one with red flowers (the dominant trait, R ) and one with white flowers (the recessive trait, r ), would be expected to produce an F 1 (first) generation with only red flowers because the allele for red flower is dominant to that of white. Using a Punnett square is a convenient method to keep track of the kinds of gametes that can be produced and the combination of gametes at fertilization. The circles along the top and side of the Punnett Square represent the possible gamete nuclei, and the squares represent the possible offspring genotypes. A white-flowered plant ( rr ) is crossed with a homozygous dominant, red-flowered plant ( RR ). Insert the proper letters indicating the genotypes of the possible gametes for the white/red flower cross in the circles, then fill in the Punnett square for all the possible genetic outcomes. The genotype(s) of the gametes of the white-flowered plant is __________ The genotype(s) of the gametes of the red-flowered plant is ____________ The genotype(s) of the plants produced by the cross is ________________ The phenotype(s) of the plants produced by the cross is _______________ A white-flowered plant is crossed with a heterozygous plant. Fill in the Punnett Square and give the genotypes and phenotypes of all the possible genetic outcomes. Possible genotypes: ____________________________________________________ Possible phenotypes: __________________________________________________

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -2 It is unlikely that every cross between two plants will produce four seeds that will grow up into four offspring every time. Rather, genetic problems like the one illustrated above allow us to predict the chances of a particular genetic outcome. Genetics is a matter of probability, the likelihood of the occurrence of a particular outcome. Consider that the probability of coming up with heads in a single toss of a coin is one chance in two, or ½. Applied to the monohybrid problem, the probability of having a certain genotype is the sum of all occurrences of that genotype. For example, the genotype Rr occurs in two of the four boxes in the Punnett Square. The probability that the genotype Rr will be produced from that particular cross is thus 2 / 4 , or 50%. What is the probability of an individual from that cross having the genotype rr ? _________ The above cross between two homozygous individuals, one with the dominant trait (red flowering plant, RR) and one with the contrasting recessive trait (white flowering plant, rr ), resulted in an F 1 (first filial) generation that is all heterozygous red flowering individuals ( Rr ). Use the Punnett Square to cross two heterozygous F 1 plants to produce the F 2 (second filial) generation. Possible genotypes: _____________________________________________________ Possible phenotypes: ____________________________________________________ In summary, the F 2 generation of a monohybrid cross with dominance will result in a: Phenotypic ratio of 3:1 Genotypic ratio of 1:2:1 This type of experimental monohybrid cross led Mendel to develop his first law of inheritance. Mendel’s first law of inheritance = Law of segregation 1) There are alternative versions of genes (alleles) that account for variation in traits 2) Each organism contains two alleles for each trait; one allele was inherited from each parent 3) The two alleles for each trait segregate (separate) during gamete production (during meiosis) 1.MONOHYBRID CROSS IN CORN MATERIALS NEEDED: - Genetic corn ears illustrating monohybrid cross (Riker Mounts) Examine the monohybrid genetic corn demonstration. They illustrate a monohybrid cross between plants producing purple kernels and ones producing yellow kernels. “P” stands for the parental generation. F 1 stands for the first offspring generation (first filial generation). If these F 1 offspring are crossed again, their offspring are the second filial generation, called F 2 . The allele for purple color is R , the allele for yellow color is r . 1. Note that all the first-generation kernels (F 1 ) in the genetic corn demonstration is purple, while the second-generation ear (F 2 ) has both purple and yellow kernels. 2. Count the numbers of purple and yellow kernels on 3 of the F 2 ear. When reduced to the lowest common denominator, is this ratio closer to 1:1, 2:1, 3:1, or 4:1? (circle one) This is called the phenotypic ratio. 3. A corncob with its many kernels represents the products of multiple instances of sexual reproduction. Each kernel represents a different cross, each arising from a different fertilization event (one egg by one sperm).

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -3 What genotype produces a purple phenotype? ________________ Which allele is dominant? ________________ What is the genotype of the yellow kernels on the F 2 ear? ___________ You are given an ear with purple kernels. How do you determine its genotype with a single cross? ____________ ___________________________________________________________________________________________ ___________________________________________________________________________________________ Table1: Number of kernels with different phenotypes Corn Ear Yellow Purple 1 2 3 Total Proportion

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity -4 TABLE 2: INCOMPLETE DOMINANCE/CODOMINANCE A) MONOHYBRID PROBLEMS WITH INCOMPLETE DOMINANCE Petunia flower color is governed by two alleles, but neither allele is truly dominant over the other. Petunias with the genotype R 1 R 1 are red-flowered, those that are heterozygous ( R 1 R 2 ) are pink, and those with the R 2 R 2 genotype are white. This is an example of incomplete dominance . (Note that superscripts are used rather than upper- and lowercase letters to describe the alleles). 1. If a white-flowered plant is crossed with a red-flowered petunia, what is the genotypic ratio of the F 1 ? _________________________________________________________ 2. What is the phenotypic ration of the F 1 ? _________________________________ 3. If two of the F 1 offspring were crossed, what phenotypes would appear in the F 2 ? _________________________________________________________________ 4. What would be the genotypic ratio of the F 2 generation? ____________________ B) MONOHYBRID PROBLEMS ILLUSTRATING CODOMINANCE Another type of monohybrid inheritance involves the expression of both phenotypes in the heterozygous situation. This is called codominance . One of the well-known examples of codominance occurs in the coat color of Shorthorn cattle. Those with reddish- gray coats (= roan) are heterozygous ( RR’ ), and result from mating between a red ( RR ) and a white ( R’R’ ) cattle. Roan cattle do not have roan-colored hairs, as would be expected with incomplete dominance, but rather appear roan because of both red and white hairs being in the same animal. Because the R and R ’ alleles are both fully expressed in the heterozygote, they are codominant. 1. If a roan Shorthorn cow is mated with a white bull, what will be the genotypic and phenotypic rations in the F 1 generation? Genotypic ratio:______________ Phenotypic ratio:________________ 2. List the parental genotypes of crosses that could produce at least some: Roan Offspring:_________________________________________________________ White offspring:______________________________________________________

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -5 3: SEX LINKED AND MULTIPLE ALLELES A) MONOHYBRID, SEX-LINKED PROBLEM–INHERITANCE OF COLOR BLINDNESS Sex is determined by chromosomes, hormones and expression of gender determining genes. Typically, in mammals, individuals containing two X chromosomes is a female, while individuals possessing an X and a Y chromosome is a male. But sex, just like any other trait in organisms does exhibit variation as determined by the genes and their expression patterns. 1. In terms of sex chromosomes, what type of gametes (ova) does a female mammal produce? ___________________ 2. What are the possible sex chromosomes in a male’s sperm? ____________________________________ 3. The gametes of which parent will determine the sex of the offspring? ________________________________ The sex chromosomes bear alleles for traits, just as other chromosomes in our bodies. Genes that occur on the sex chromosomes are said to be sex-linked . More specifically, the genes present on the X chromosome are said to be X- linked, those genes found on the Y chromosome are said to be Y-linked. There are many more genes present on the X chromosome than are found in the Y chromosome. The Y chromosome is smaller than its homologue, the X chromosome. Consequently, some of the loci present on the X chromosome are absent on the Y chromosome. In humans, the gene for color vision is located on the X but it is absent from the Y chromosome (i.e. color vision is X-linked). The figure to the right illustrates the appearance of duplicated sex chromosomes, each consisting of two sister chromatids, and the location of a sex- linked trait, N. Normal color vision ( X N ) is dominant over color blindness ( X n ). Suppose a color-blind man ( X n Y) father’s children of a woman with the genotype ( X N X N ). 1. What proportion of daughters would be color-blind? _____________________ 2. What proportion of sons would be color-blind? _________________________ One of the daughters from the above problem marries a color-blind man. 3. What proportion of their sons will be color-blind? _________________________ (Hint: Another way to think of this is to ask what the chances are that their sons will be color- blind). 5. Explain how a color-blind daughter might result from this couple. ___________________________________________________________________ ___________________________________________________________________

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -6 B) MULTIPLE ALLELES – THE ABO and Rh BLOOD GROUP SYSTEM Last month the molecular basis of a rare fatal hemolytic condition in newborns was published in the Journal Blood ( https://ashpublications.org/blood/article-abstract/doi/10.1182/blood.2022016504/486664/Missense-mutations-in- PIEZO1-encoding-the-Piezo1?redirectedFrom=fulltext ). This is the 44 th blood group that has been discovered till date: See https://www.sciencealert.com/discovery-of-a- new-rare-blood-type-could-save-the-lives-of-future-newborns . We will be studying the familiar ABO and Rh blood group in this lab. ABO Blood group Karl Landsteiner was investigating the effects of mixing red blood cells (RBC, erythrocytes) from one person with serum from another and noted some RBCs exhibited clumping or agglutination. This led to his discovery of the ABO blood group in humans, and he published his landmark paper on blood types in 1901 explaining how antibodies in serum reacting with antigens on the surface of RBCs caused the agglutination. He worked with his students and others and later identified the AB blood group as well as Rh factors and also showed that a virus was responsible for Polio. He was awarded the Nobel Prize in Physiology or Medicine in 1930. Current research has further extended these findings and today we know the molecular basis and genes responsible for these blood types. The AB antigens are the result of enzymes adding specific carbohydrate residues to the H antigen on the surface of RBCs. There are two genes that determine the ABO blood group. The FUT1 gene ( FU cosyl T ransferase) on Chromosome 19 catalyzes the transfer of L-fucose to a galactose residue leading to H antigen synthesis on the surface of RBCs. This gene can be either homozygous dominant (HH) or heterozygous (Hh) or a rare hh genotype which is generally present in only about 0.0004% (~ 4 per million) of the human population, known as Bombay blood type. This blood type has no H antigen on the surface of red blood cells. The hh individuals will have the H antibody in their blood and hence can only receive blood from others with the same hh genotype. The ABO gene is on Chromosome 9, made up of seven exons and in this ABO blood group system there are at least three allelic forms: I A , I B , or I (o) . Alleles I A and I B are codominant, while the allele i is recessive. From: https://www.jstage.jst.go.jp/article/jmi/ 55/3,4/55_3,4_174/_pdf/char/en As shown in the figure on the left, there are four amino acid differences between the allele A ( Arg- Glu-Leu-Gly) and allele B ( Gly-Ser-Met-Ala) in Exon 7 creating two different enzymes. The i (O)allele has a base deletion in exon 6.

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity -7 The A allele encodes an acetylgaltosaminytransferase, which modifies the H antigen into the A antigen by adding the sugar, N-acetylgalactosamine (GalNAc) to it, and the B allele encodes a galactosyltransferase which modifies the H antigen into the B antigen by adding a galactose to it. The antigenic specificity is due to the addition of these carbohydrate modifications to antigen H. A deletion in the i (o) allele in exon 6 results in a frame shift mutation creating an immature stop codon resulting in a truncated protein which is nonfunctional and hence no carbohydrates are added to the H antigen. It therefore remains unmodified leading to the O blood group. In individuals who are typed AB, they are heterozygous and have both A and B alleles, the H antigen may be modified by the addition of either GalNAc or galactose. The Rh blood type On chromosome 1 two genes, consisting of ten exons each encode the products of the most polymorphic of the blood groups making up these Rh antigens. There are at least 45 different Rh antigens are known and the deletion of the RHD gene results in Rh negative (Rh-) blood type. This phenotype is the result of dominant alleles, with either homozygous dominant or heterozygous genotypes (+/+ or +/-) for those who are Rh positive and homozygous recessive (-/-) genotype for Rh negative individuals. The Rh antigens are highly immunogenic, but in the absence of the Rh antigen, no antibody is present. If the blood of someone who is Rh negative is exposed to an Rh positive blood, their blood will soon exhibit a reaction because their immune system will very quickly start producing antibodies. This is the reason why apart from matching the ABO blood type, the Rh blood group should also be matched for blood transfusions. Experiment to identify blood type: In today’s lab you will identify both the ABO as well as Rh blood type of four samples. In this lab experiment samples used are simulated and not real blood or antisera and are safe to handle. The alleles I A and I B code for production of proteins called Antigen A and Antigen B on the surface of red blood cells, and i doesn’t code for an antigen (no antigen = “O”). The immune system produces antibodies to antigens in order to protect an organism from foreign invaders such as bacteria and viruses. An organism does not normally generate antibodies to antigens found on its own cells, because that would destroy the cells. Therefore, a person with blood type A (Antigen A) will not produce Anti-A antibody, but she will produce Anti-B antibody. If that woman then needs a blood transfusion, she cannot receive type B blood (Antigen B), because her Anti-B antibody will destroy the donated blood cells. Using the above information, complete this table: Table2: The ABO Blood Groups Blood Type (Phenotype) Antigen Present Antibody Present. There can be more than one Genotype (s) A A Anti-B I A I A or I A i B AB O Blood Type (Phenotype) Antigen Present Antibody Present. Genotype Rh + Rh – (not exposed to Rh+ blood) Rh – (exposed to Rh+ blood)

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -8 Procedure to identify blood type: Place four of the white plastic plates on the table and label them as shown below with a permanent marker with a number and appropriate labels for each of the wells, if they are not already labelled. If there are four students in your group, each student may take one sample tube and do the reaction for a plate. 1. Using the dropper vial, place a drop of the Sample 1 synthetic blood sample in each well of the blood typing slide marked 1. Close the cap on the dropper vial. To prevent cross contamination, always close the cap on one vial before opening the next vial. 2. Add a drop of synthetic anti-A serum (blue) to well A . Close the cap. 3. Add a drop of synthetic anti-B serum (yellow) to well B . Close the cap. 4. Add a drop of synthetic anti-Rh serum (clear) to well Rh . Close the cap. 5. Using a different-colored mixing stick (or toothpick) for each well (blue for anti-A, yellow for anti-B, white for anti- Rh), gently mix the synthetic blood and antiserum drops for 30 seconds. Remember to use a new mixing stick for each sample to avoid contamination of your samples. 6. Examine the resulting films of liquid mixture in the well. If a film is uniform in appearance, there is no agglutination. If the sample is granular, agglutination has occurred. Note: Since this is simulated blood, the reaction only approximates agglutination that would occur if we used real blood and antisera and hence the Rh reaction in this sample may just turn sticky if it is a positive reaction. 7. Fill in the results for Sample 1 in the following Data Table, answering yes or no as to whether agglutination occurred with each antiserum. 8. Repeat steps 1 through 7 using synthetic blood Sample bottles 2, 3, 4 and plates 2, 3, and 4. 9. Please thoroughly wash, rinse the blood typing slide and mixing sticks in water and wipe them before returning them back to the plastic container. Results Blood Sample 1 Blood Sample 2 Blood Sample 3 Blood Sample 4 Anti-A serum Anti-B serum Anti-Rh serum

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -9 Blood type = 1. Is it possible for a child of blood type O to be produced by two AB parents? ___________________________ Explain: __________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 2. In a case of disputed paternity, the child is type O, the mother type A. Could an individual of the following blood types be the father? O ___________________________________ A _____________________________________ B ___________________________________ AB ____________________________________ 3. A person who has the Bombay blood type was incorrectly identified as having the O- blood group, but he almost died after a blood transfusion after a surgery. Explain why he was misidentified as having O type blood _________________________________________________________________________________________________ Why did he exhibit such a bad reaction after the transfusion? __________________________________________________________________________________________________ 4. How many alleles are there for the H gene? _______________________. How many alleles are there for the ABO gene? ______________________. 5. Why would the genes for ABO blood type and Rh blood type exhibit independent assortment? __________________________________________________________________________________________________ 6. Is it possible for a Rh- mother to have a child who could be Rh+? Explain: __________________________________________________________________________________________________

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity -10 4: DIHYBRID INHERITANCE A dihybrid cross is a cross between two individuals that differ in two traits. For example, imagine a cross of two homozygous individuals, one with two dominant traits (red, large flowers, RRLL) and one with the contrasting recessive traits (white, small flowers, rrll ). This cross would result in an F 1 generation that is all heterozygous red, large flowers. If the F 1 generation is crossed to produce the F 2 generation, the results are a little different than in a monohybrid cross. In the monohybrid F 2 there were only two phenotypes. In a dihybrid cross, there are four phenotypes - homozygous and heterozygous red, large flowered plants ( R_L_), red small flowered plants ( R_ll ), white large flowered plants ( rrLl ) and white small flowered plants ( rrll ) In summary, the F2 generation of a dihybrid cross, with dominance in the two traits will result in a: Phenotypic ratio of 9:3:3:1 Genotypic ratio of 1:2:2:1:4:1:2:2:1 This type of experimental dihybrid cross led to the Mendel’s second low of inheritance. Mendel’s second law of inheritance = Law of Independent Assortment Traits are inherited independent from each other. Independent assortment occurs during Anaphase of Meiosis I. DIHYBRID PROBLEM WITH CORN SNAKES Corn snakes ( Elaphe guttata ) are popular in the pet trade, and a number of color and pattern morphs are recognized. Corn snakes have two main colors - red and black. A normal corn snake’s genotype (“wild-type”) can be represented as " R_B_ ". The dominant allele R causes production of the red pigment. Snakes that are homozygous recessive ( rr ) are unable to make red pigment. They are known as anerythristic. The dominant allele B causes the production of black pigment. Corn snakes that are homozygous recessive ( bb ) are unable to make black pigment and are referred to as amelanistic. Lastly, corn snakes that make neither read nor black are called snow corns. In the table below, list all possible genotypes the different color morphs of Corn snakes: Table3: Possible genotypes for color and pattern morphs of Corn Snakes wild type (red and black) amelanistic (red but lacks black) anerythristic (lacks red but has black) snow (neither red nor black) __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________ __________________

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -11 A snake is heterozygous for both red and black coloration. What is the genotype of such a snake? _________________ 1. List the possible genotypes of that snake’s gametes? ________________________________________________ (Remember that the principle of independent assortment states that genes on different (nonhomologous) chromosomes are separated out independently of one another during meiosis. The occurrence of an allele for red coloration in a gamete has no bearing on which allele for black coloration will occur in that same gamete). 2. Two snakes heterozygous for both colors (red and black) are bred. Use a Punnett Square to determine the offspring’s genotypes. List the possible phenotypes that may result from such a cross: ____________________________________________________ ____________________________________________________ What is the probability of an offspring having the following genotype: RrBb _________ RRBb _________ rrBB _________ To extend the probability discussion, let’s consider flipping a coin by asking the question “What is the probability of flipping heads twice in a row?” The chance of flipping heads the first time is ½. The chance (probability) that we’ll flip heads twice in a row is ½ x ½ = ¼. The probability that we could flip heads 3 times in a row is ½ x ½ x ½ = 1 / 8 . 3. If you would breed two corn snakes that are both heterozygous for red and black coloration, what is the probability that three baby snakes in the clutch will have the genotype rrbb ? _________________________________________________________ 4. What is the genotype of the F 1 generation if one snake is homozygous for both red and black color, but the other snake is a “snow” (i.e., homozygous recessive for red and black)? _________________________________________________________ What is the phenotype of these snakes? ________________________ 5: HUMAN TRAITS WITH MENDENLIAN INHERITANCE Many human traits are complex and involve many genes or interactions between genes. For example, hair color is determined by at least 4 genes, each coding for the production of melanin, a brown pigment. Because the effect of these genes is cumulative, hair color can range from blond (little melanin) to very dark brown (much melanin). Below is a list of human traits that exhibit Mendelian inheritance. Tongue Rolling - Attempt to roll your tongue into a U-shape, in which the sides of your tongue are curled upwards. Tongue rollers carry a dominant allele R . Non-tongue rollers are homozygous recessive ( rr ).

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -12 Widow’s Peak - A dominant allele W causes the hairline to form a distinct downward point in the center of the forehead. If there is no downward point of the hairline, the person is homozygous recessive ( ww ). Earlobe Attachment - The inheritance of a dominant allele E results in the free (unattached) earlobe. If the lobe is attached directly to the head, the individual is homozygous recessive ( ee ). Hitchhiker's Thumb - Some individuals can bend the last joint of the thumb backwards at about a 45-degree angle. These individuals are homozygous for a recessive allele, hh. Those who cannot bend at least one thumb backwards about 45 degrees, are carrying the dominant allele, H . Mid-digital Hair - The presence of hair on the middle segment of the fingers is caused by a dominant allele, M . The homozygous recessive condition, mm, results in the lack or absence of hair on the middle segments of the fingers. Examine your hands closely since the hairs may be small in length and light in color. Dimpled Chin - A dimpled chin is caused by a dominant allele, C , a normal chin is homozygous recessive ( cc ). Big Toe Length - The length of the big toe is governed by the dominant allele, T. The inheritance of the homozygous recessive condition ( tt ) results in the big toe being longer than or equal to the second toe. Red-Green Colorblindness - The allele for this trait is located on the X chromosome and is thus said to be sex- linked. If you see colors normally, you carry the dominant allele for this trait (either X B X B or X B X b for females and X B Y for males). If, on the other hand, you are red-green colorblind, you carry the recessive allele. Colorblind males are X b Y, and colorblind females are X b X b . Homework: Based on what you learnt about Monohybrid and Dihybrid crosses, and different types of Mendelian inheritance, complete the homework worksheet upload it in Canvas. You can complete the homework on MS Word and upload it in Canvas or you can complete the homework on paper copy, take a picture and upload the picture to Canvas. This homework is worth 10 points.

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity -13 6: How does the Rapid Covid-19 Test Work? On December 12, 2019, the Chinese reported the first cases of an illness in the city of Wuhan. WHO on January 10, 2020, designated it as a ‘2019 novel corona virus or 2019-nCoV’ to refer to this disease outbreak and on the same day Chinese biologists published the sequence ( MN908947) of the pathogen causing the disease in GenBank. On February 11, 2020, the International Committee on Taxonomy named this novel virus, as Severe acute respiratory syndrome coronavirus 2, shortened to SARS- CoV-2 and WHO officially named the disease caused by this SARS-CoV2 as Covid-19. The disease quickly began to spread to all countries, with a fatality rate more than 20 times that of the flu and with no effective treatment or vaccine, several countries went into lockdown and less than three months after the illness was first identified in China the virus started spreading in the US and with a concurrent increase in fatality the US officially went into lockdown mode on March 15, 2020. Covid-19 soon became a leading cause of death worldwide and especially in the US. Based on CDC data it even temporally superseded the death rates of heart disease and cancer in the US with two spikes in 2020, with deaths as much as 4000 per day here. For the first time, using the RNA sequence this corona virus uses to make the spike protein on the outer envelope, mRNA and DNA vaccines were developed by Pfizer, Moderna and Johnson & Johnson within a year and became available by mid-December 2020 became available to the general public. Still, with all the precautions of a lockdown, social distancing and advise to wear a face mask the death toll from covid-19 has exceeded over a million and is currently at 1,125,366 individuals who have died of this disease. Please see: https://www.cdc.gov/museum/timeline/covid19.html#:~:text=March%2015%2C%202020,the%20spread%20of %20COVID%2D19 for a good summary of events of the Covid-19 pandemic. Hence the need for quick home tests to identify those who become infected with covid for their own heath as well as to protect those who are more vulnerable to this pathogen was quickly realized as necessary in addition to the more sensitive but more expensive tests such as RT-PCR that needed specialized equipment. Development of Rapid Covid tests Several cost effective covid tests became available with varying levels of sensitivity and accuracy and was approved by FDA starting mid-December, 2020, but the most commonly used home test for covid-19 and is manufactured by several companies such as Abbott uses the lateral flow method which is a qualitative calorimetric assay based on visualizing a color change in the test and control line.

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -14 How is the Covid-19 Rapid test strip constructed? Figure 1 : The strip consists of a space to add your nasal swab sample and next to it, is an area (1) containing mobile primary antibodies to the SARS-CoV2 antigen labeled with nanoparticles. Then in area (2) are unlabeled primary antibodies that are immobile and further down in area (3) are immobile secondary antibodies, which are antibodies to the primary antibodies. How does the Covid-19 test strip work? The liquid from the nasal swab sample is wicked into the strip after it is applied and travels towards the antibodies by lateral flow. 1) If a test is positive , then the labeled antibodies with antigens to the SARS-CoV2 virus will bind together and flow towards the immobile antibodies and bind to both the antibodies in the test line as well as to the secondary antibodies in the control line as shown in Figure 2.

Foundations of Biological Sciences I - Lab Manual Principles of Heredity -15 2) If a test is negative for covid, the mobile labeled antibodies only bind to the secondary antibodies which are antibodies to the SARS-CoV2 antibodies as shown in Figure 3. All figures above are adapted from Lab exercise: In today’s lab you will do a simple setup to understand how lateral flow is used to detect SARS-Cov2. Cut a two inch strip of Magic white wipe and a width of about half an inch. Press a plastic ruler on its edge across this white substrate. Keeping the ruler in place add 4-5 drops of Solution A on one side of the white strip as shown in Figure 4. This will represent the antibody. We will only show the color change for one line and assume it is the test line. Add 2ml of Solution B , to represent the solution with the virus and hence the antigens into a small plastic petri dish and slightly tilt the dish before placing the edge (that is further from where you added Solution A) of the prepared test strip into it. Stand the strip vertically for ~ 30 seconds and then place the strip horizontally at an angle, resting the strip against the lip of the plastic petri dish. Watch for the color change.

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Foundations of Biological Sciences I - Lab Manual Principles of Heredity -16 Questions: Circle the correct answer. 1) What type of antibodies are present in Area 1 of the test strip? a) Primary antibodies to SARS-CoV2 with nanoparticles conjugated to it. b) Primary antibodies to SARS-CoV2 without nanoparticles conjugated to it. c) Secondary antibodies that are antibodies to the primary antibodies to SAR-CoV2. 2) What type of antibodies are present in Area 2 of the test strip? a) Primary antibodies to SARS-CoV2 with nanoparticles conjugated to it. b) Primary antibodies to SARS-CoV2 without nanoparticles conjugated to it. c) Secondary antibodies that are antibodies to the primary antibodies to SAR-CoV2. 3) What type of antibodies are present in Area 3 of the test strip? a) Primary antibodies to SARS-CoV2 with nanoparticles conjugated to it. b) Primary antibodies to SARS-CoV2 without nanoparticles conjugated to it. c) Secondary antibodies that are antibodies to the primary antibodies to SAR-CoV2. 4) Which of the following are mobile or immobile in the covid-19 test strip? a) Primary antibodies to SARS-CoV2 with nanoparticles conjugated to it. Mobile \ Immobile b) Primary antibodies to SARS-CoV2 without nanoparticles conjugated to it. Mobile \ Immobile c) Secondary antibodies that are antibodies to the primary antibodies to SAR-CoV2. Mobile \ Immobile 5) Briefly explain how both the test line as well as the control line becomes visible if a test is positive for covid-19.

Lab 8 - Heredity and Genetics of Blood type and the rapid antigen test

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