Problem E.10b
The frequencies of the four alleles contributed to the child by possible father F1 in Problem 7 are 0.18, 0.23, 0.13, and 0.14. Make a statement about the possible paternity of F1 based on this analysis.
Problem E.11a
In an inheritance case, a man has died leaving his estate to be divided equally between 'his wife and his offspring.' His wife (M) has an adult daughter (D), and they argue that they should split the estate equally. As a young couple, however, the man and his wife had a son that they gave up for adoption. Two men have appeared, each claiming to be the son of the couple and therefore entitled to a one-third share of the estate. The accompanying illustration shows the results of DNA analysis for five genes for the mother (M), her daughter (D), and the two claimants (S1 and S2). How many nonmaternal DNA bands are shared by D and S1? By D and S2?
Problem E.12a
Three independently assorting STR markers (A, B, and C) are used to assess the paternity of a colt recently born to a quarter horse mare. Blood samples are drawn from the mare, her colt, and three possible male sires (S₁, S₂, and S₃). DNA at each marker locus is amplified by PCR, and a DNA electrophoresis gel is run for each marker. Amplified DNA bands are visualized in each gel by ethidium bromide staining. Gel results are shown below for each marker. Evaluate the data and determine if any of the potential sires can be excluded. Explain the basis of exclusion, if any, in each case.
Problem E.12b
Three independently assorting STR markers (A, B, and C) are used to assess the paternity of a colt recently born to a quarter horse mare. Blood samples are drawn from the mare, her colt, and three possible male sires (S₁, S₂, and S₃). DNA at each marker locus is amplified by PCR, and a DNA electrophoresis gel is run for each marker. Amplified DNA bands are visualized in each gel by ethidium bromide staining. Gel results are shown below for each marker. Calculate the PI and CPI based on these STR markers, using the following population frequencies: A₁₂ = 0.12, A₁₀ = 0.18; B₁₈ = 0.08, B₁₂ = 0.17; C₁₆ = 0.11, C₁₄ = 0.20.
Problem E.6a
A victim of murder is found to have scrapings containing skin cells under several of her fingernails. Genetic analysis confirms that the DNA isolated from these cells came from the same individual and does not match the DNA of the victim. The results shown below are for six CODIS STR markers from the crime scene DNA (from under the victim's fingernails and presumed to be the murderer's), and from three suspects (A, B, and C) who have been detained for questioning about the murder. Do the STR results exclude any of the three suspects? Explain.
Problem E.6b
A victim of murder is found to have scrapings containing skin cells under several of her fingernails. Genetic analysis confirms that the DNA isolated from these cells came from the same individual and does not match the DNA of the victim. The results shown below are for six CODIS STR markers from the crime scene DNA (from under the victim's fingernails and presumed to be the murderer's), and from three suspects (A, B, and C) who have been detained for questioning about the murder. Is there a failure to exclude any of the suspects? Explain.
Problem E.7a
The results shown are from a DNA test for four genes used in a paternity identification case. DNA for the mother (M) and her child (C) are shown along with DNA from two possible fathers, F1 and F2. In the 'C' column, label the DNA bands contributed by the mother with 'M' and the DNA bands contributed by the father with 'F.'
Problem E.7b
The results shown are from a DNA test for four genes used in a paternity identification case. DNA for the mother (M) and her child (C) are shown along with DNA from two possible fathers, F1 and F2. Based on the exclusion principle, is either man excluded as the possible father? Explain.
Problem E.7c
The results shown are from a DNA test for four genes used in a paternity identification case. DNA for the mother (M) and her child (C) are shown along with DNA from two possible fathers, F1 and F2. What can you conclude based on the DNA results available?
Problem 1
What purpose do the bla and lacZ genes serve in the plasmid vector pUC18?
Problem 2a
The human genome is 3×10⁹ bp in length.
How many fragments would be predicted to result from the complete digestion of the human genome with the following enzymes: Sau3A (˘GATC), BamHI (G˘GATCC), EcoRI (G˘AATTC), and NotI (GC˘GGCCGC)?
Problem 2b
The human genome is 3×10⁹ bp in length.
How would your initial answer change if you knew that the average GC content of the human genome was 40%?
Problem 3
Ligase catalyzes a reaction between the 5′ phosphate and the 3′ hydroxyl groups at the ends of DNA molecules. The enzyme calf intestinal phosphatase catalyzes the removal of the 5′5′ phosphate from DNA molecules. What would be the consequence of treating a cloning vector, before ligation, with calf intestinal phosphatase?
Problem 4a
You have constructed four different libraries: a genomic library made from DNA isolated from human brain tissue, a genomic library made from DNA isolated from human muscle tissue, a human brain cDNA library, and a human muscle cDNA library.
Which of these would have the greatest diversity of sequences?
Problem 4b
You have constructed four different libraries: a genomic library made from DNA isolated from human brain tissue, a genomic library made from DNA isolated from human muscle tissue, a human brain cDNA library, and a human muscle cDNA library.
Would the sequences contained in each library be expected to overlap completely, partially, or not at all with the sequences present in each of the other libraries?
Problem 5a
Using the genomic libraries, you wish to clone the human gene encoding myostatin, which is expressed only in muscle cells.
Assuming the human genome is 3x10⁹ bp and that the average insert size in the genomic libraries is 100 kb, how frequently will a clone representing myostatin be found in the genomic library made from muscle?
Problem 5b
Using the genomic libraries, you wish to clone the human gene encoding myostatin, which is expressed only in muscle cells.
How frequently will a clone representing myostatin be found in the genomic library made from brain?
Problem 5c
Using the genomic libraries, you wish to clone the human gene encoding myostatin, which is expressed only in muscle cells.
How frequently will a clone representing myostatin be found in the cDNA library made from muscle?
Problem 5d
Using the genomic libraries, you wish to clone the human gene encoding myostatin, which is expressed only in muscle cells.
How frequently will a clone representing myostatin be found in the cDNA library made from brain?
Problem 6
The human genome is 3×10⁹ bp. You wish to design a primer to amplify a specific gene in the genome. In general, what length of oligonucleotide would be sufficient to amplify a single unique sequence? To simplify your calculation, assume that all bases occur with an equal frequency.
Problem 7a
Using animal models of human diseases can lead to insights into the cellular and genetic bases of the diseases. Duchenne muscular dystrophy (DMD) is the consequence of an X-linked recessive allele.
How would you make a mouse model of DMD?
Problem 7b
Using animal models of human diseases can lead to insights into the cellular and genetic bases of the diseases. Duchenne muscular dystrophy (DMD) is the consequence of an X-linked recessive allele.
How would you make a Drosophila model of DMD?
- Compare methods for constructing homologous recombinant transgenic mice and yeast.
Problem 8
- Chimeric gene-fusion products can be used for medical or industrial purposes. One idea is to produce biological therapeutics for human medical use in animals from which the products can be easily harvested—in the milk of sheep or cattle, for example. Outline how you would produce human insulin in the milk of sheep.
Problem 9
- Why are diseases of the blood simpler targets for treatment by gene therapy than are many other genetic diseases?
Problem 10
Problem 11
Injection of double-stranded RNA can lead to gene silencing by degradation of RNA molecules complementary to either strand of the dsRNA. Could RNAi be used in gene therapy for a defect caused by a recessive allele? A dominant allele? If so, what might be the major obstacle to using RNAi as a therapeutic agent?
Problem 12
Compare and contrast methods for making transgenic plants and transgenic Drosophila.
- It is often desirable to insert cDNAs into a cloning vector in such a way that all the cDNA clones will have the same orientation with respect to the sequences of the plasmid. This is referred to as directional cloning. Outline how you would directionally clone a cDNA library in the plasmid vector pUC18.
Problem 13
- A major advance in the 1980s was the development of technology to synthesize short oligonucleotides. This work both facilitated DNA sequencing and led to the advent of the development of PCR. Recently, rapid advances have occurred in the technology to chemically synthesize DNA, and sequences up to 10 kb are now readily produced. As this process becomes more economical, how will it affect the gene-cloning approaches outlined in this chapter? In other words, what types of techniques does this new technology have potential to supplant, and what techniques will not be affected by it?
Problem 14
Problem 15a
The bacteriophage lambda genome can exist in either a linear form or a circular form.
How many fragments will be formed by restriction enzyme digestion with XhoI alone, with XbaI alone, and with both XhoI and XbaI in the linear and circular forms of the lambda genome?
Ch. 15 - Recombinant DNA Technology and Its Applications