Chapter 10 Flash Cards

*10.40 The frequency of individuals in a population with two different alleles at a DNA marker is called the marker's heterozygosity. Why would an STR DNA marker with nine known alleles and a heterozygosity of 0.79 be more useful for mapping and DNA fingerprinting studies than a nearby STR having three alleles and a heterozygosity of 0.20?

Because if STR has few alleles and low heterozygosity many individuals in a population will share the same STR genotype and many individuals in the population who by chance alone will share the same genotype as a test subject and the STR will not be very useful for DNA fingerprinting studies.

*10.4 Suppose you have cloned a eukaryotic cDNA and want to express the protein it encodes in E. coli. What type of vector would you use, and what features must this vector have? How would this vector need to be modified to express the protein in a mammalian tissue culture cell?

An expression vector which have the signals necessary for DNA inserts to be transcribed and for these transcripts to be translated. In eukaryotes a promoter is needed and a poly (A) site should be downstream from where the cDNA is inserted.

10.14 A molecular genetics research laboratory is working to develop a mouse model for bovine spongiform encephalopathy (BSE) ("mad cow") disease, which is caused by misfolding of the prion protein. As part of their investigation, they want to investigate the structure of the gene for the prion protein in mice. They have a mouse genomic DNA library made in a BAC vector and a 2.1-kb long cDNA for the gene. List the steps they should take to screen the BAC library with the cDNA probe.

1. Determine how many BACs you need to screen and screen about 35,000 BACs to be 90% sure of obtaining a BAC clone with the gene. 2. Plate E.coli cells harboring the BACs onto bacterial plates wirh growth medium that selects for the presence of the BACs. 3. Pick either individual BAC containing colonies, grow them up in microtiter plates and array them into grids on membranes. 4. Remove the membrane filters with the colonies from their culture dishes, lyse the bacteria that are growing on then and allow the denatured DNA to bind the filter. 5. Make a probe by using the cDNA template and random-primer. 6. Incubate the probe with the DNA filters in a heat-sealable plastic bag and allow the probe to hybridize to complementary BAC sequences that are bound to the filters. 7. Wash the filters free of unbound probe and then detect the location of the hybridize probe using autoradiography for a radioactively labeled probe or chemiluminescent detection for a nonradioactive labeled probe. 8. Pick the BAC colonies that have sequences complementary to the probe based on the locations of the hybridization signal.

10.15 A scientist has carried out extensive studies on the mouse enzyme phosphofructokinase. He has purified the enzyme and studied its biochemical and physical properties. As part of these studies, he raised antibodies against the purified enzyme. What steps should he take to clone a cDNA for this enzyme?

1. Prepare cDNAs using mRNA isolate from a mouse tissue where phosphofructokinase is abundant. 2. Clone the cDNAs into a plasmid expression vector using linkers. 3. Transform the E.coli with cDNA clones, and plate the bacteria on a medium that selects for the presence of plasmid. 4. Transfer individual colonies to microtiter dishes and then grow and store the bacteria. 5. Print colonies to a membrane filter, and grown then on the medium to express the protein products of the cloned cDNA. 6. Remove the filters from their medium, lyse the cells in sity and allow the protein products to be bound to the filters. 7. Label the antibodies with radioactivity, and incubate the labeled antibodies with the membrane filters. 8. Wash off unbound antibodies with radioactivity, and incubate the label antibodies with the membrane. 9. Pick the bacterial colonies that align with the radioactive signal, grow them up, and purify the cDNA bearing plasmids from these colonies.

*10.6 You have inserted human insulin cDNA in the cloning vector pBluescript II (described in Figure 8.4, p. 176) and transformed the clone into E. coli, but insulin was not expressed. Propose several hypotheses to explain why not.

1st- in order for insulin to be expressed, it must be inserted in the correct reading frame for premature termination of translation does not occur and the correct peptide is produced. 2nd- it would be important to ensure that only the ORF (open reading frame) of the insulin gene is properly inserted into the polylinker of the pBluescrpit II vector. 3rd- a complement copy of the human mRNA transcript for insulin may be used and not just the ORF. 4th- the cDNA may encode a protein that is posttranslational processed in eukaryotic cells to become human insulin.

*10.36 For rare genetic disorders that have only one mutant allele, genetic tests can be tailored to detect the mutant and normal alleles specifically. However, for more prevalent genetic disorders, such as anemia caused by mutations in - and -globin, Duchenne muscular dystrophy caused by mutations in the dystrophin gene, and cystic fibrosis caused by mutations in CFTR, there are many different alleles at one gene that can lead to different disease phenotypes. These diseases present a challenge to genetic testing because, for these diseases, a genetic test that identifies only a single type of DNA change is inadequate. How can this challenge be overcome?

By evaluating the presence of a defined set of alleles to use reverse ASO hybridization and multiplex PCR.

*10.18 It is 3 a.m. Your best friend has awakened you with yet another grandiose scheme. He has spent the last two years purifying a tiny amount of a potent modulator of the immune response. He believes that this protein, by stimulating the immune system, could be the ultimate cure for the common cold. Tonight, he has finally been able to obtain the sequence of the first seven amino acids at the N-terminus of the protein: Met-Phe-Tyr-Trp-Met- Ile-Gly-Tyr. He wants your help in cloning a cDNA for the gene so that he can express large amounts of the protein and undertake further testing of its properties. After you drag yourself out of bed and ponder the sequence for a while, what steps do you propose to take to obtain a cDNA for this gene?

Compare the amino acid sequence to the genetic code, and design a "guessmer" a set of oligonucleotides that could code for this sequence.

10.10 Restriction endonucleases are used to construct restriction maps of linear or circular pieces of DNA. The DNA usually is produced in large amounts by recombinant DNA techniques. Generating restriction maps is like putting the pieces of a jigsaw puzzle together. Suppose we have a circular piece of double-stranded DNA that has a length of 5,000 bp. If this DNA is digested completely with restriction enzyme I, four DNA fragments are generated: fragment a is 2,000 bp, b is 1,400 bp, c is 900 bp, and d is 700 bp. If, instead, the DNA is incubated with the enzyme for a short time, the result is partial digestion of the DNA: not every restriction enzyme site in every DNA molecule will be cut by the enzyme, and all possible combinations of adjacent fragments can be produced. From a partial digestion experiment of this type, fragments of DNA were produced from the circular piece of DNA that contained the following combinations of the above fragments: a-d-b, d-a-c, c-b-d, a-c, d-a, d-b, and b-c. Lastly, after digesting the original circular DNA to completion with restriction enzyme I, the DNA fragments are treated with restriction enzyme II under conditions conducive to complete digestion. The resulting fragments are 1,400, 1,200, 900, 800, 400, and 300 bp. Analyze all the data to locate the restriction enzyme sites as accurately as possible.

Consider that if the enzyme cuts in a circular molecule once, it will produce 1 fragment. If it cuts twice, it will produce 2 fragments. In this example 4 fragments are produced when enzyme I completely cleaves the plasmid, enzyme I must cut the plasmid at 4 sites. A partial digestion occurs when not all 4 sites are cut. d-a-c fragment was released when 2 cuts were made at sites flanking fragment b. a-d-b was released when cuts were made at c, and c-b-d was released when cuts were made at a. Thus a is next to c, d is next to a, b is next to c, and d is next to b. c-a-d-b is the order of the fragments in the plasmid. (SEE SOLUTIONS MANUEL FOR FURTHER EXPLANATION AND DATA)

10.41 What is DNA fingerprinting and what different types of DNA markers are used in DNA fingerprinting?How could this method be used to establish parentage?How is it used in forensic science laboratories?

DNA fingerprinting is the characterization of an individual in terms of the set of DNA markers the person has. DNA markers are RFLPs, STRs and VNTRs. DNA fingerprinting is used to match or exclude the DNA fingerprinting of a suspected individual with the DNA fingerprinting provided by physics evidence gathered at the scene of a crime.

*10.30 Chapter 9 presented a description of how DNA microarray analysis was used to characterize changes to the transcriptome during yeast sporulation. That analysis found that more than 1,000 yeast genes showed significant changes in mRNA levels during sporulation, identified at least seven distinct temporal patterns of gene induction, and provided insights into the functions of many orphan genes. It is important to confirm findings from microarray analyses using independent methods. How would you confirm independently that three orphan genes display altered expression during yeast sporulation?

Design primers so that you can use PCR to amplify a segment of each orphan gene. Then prepare RNA from yeast at sequential stages of sporulation and use reserve transcriptase to reveserce transcribe each RNA sample into cDNA. Then quantify the amount of each genes cDNA in the different cDNA preparation using real time PCR.

10.39 A research team interested in social behavior has been studying different populations of laboratory rats. By using a selective breeding strategy, they have developed two populations of rats that differ markedly in their behavior: one population is abnormally calm and placid, while the second population is hyperactive, nervous, and easily startled. Biochemical analyses of brains from each population reveal different levels of a catecholamine neurotransmitter, a molecule used by neurons to communicate with each other. Relative to normal rats, the hyperactive population has increased levels while the calm population has decreased levels. Based on these results, the researchers have hypothesized that the behavioral and biochemical differences in the two populations are caused by variations in a gene that encodes an enzyme used in the synthesis of the catecholamine. Suppose you have a set of SNPs that are distributed throughout the rat genomic region containing this gene, including its promoter, coding region, enhancers, and silencers. How could you use these SNPs to test this hypothesis?

I t can be evaluates by sequencing the gene in representative individuals from each rat population.

10.48 In 1990, the first human gene therapy experiment on a patient with adenosine deaminase deficiency was done. Patients who are homozygous for a mutant gene for this enzyme have defective immune systems and risk death from diseases as simple as a common cold. Which cells were involved, and how were they engineered?

In the gene therapy experiment, T cells were isolated from the patient and a viral vector was used to introduce the normal adenosine deaminase gene.

*10.26 A scientist is interested in understanding the physiological basis of alcoholism. She hypothesizes that the levels of the enzyme alcohol dehydrogenase, which is involved in the degradation of ethanol, are increased in individuals who routinely consume alcohol. She develops a rat model system to test this hypothesis. What steps should she take to determine if the transcription of the gene for alcohol dehydrogenase is increased in the livers of rats who are fed alcohol chronically compared to a control, abstinent population?

Isolate RNA from the livers of the alcohol-fed and control rats. Measure the levels of mRNA for alcohol dehydrogenase by ether separating the RNAby size using gel electrophoresis, preparing a northern blot, and hybridizing it with a probe made by labeling a cDNA for the alcohol dehydrogenase gene or by using TR-PCR or real time quantitative PCR.

10.32 What is meant by humanization, and how is it used to evaluate candidate drugs for treating a disease?

It is the process by which a gene from a model experimental organism such as a mouse is modified to be more like its human homolog. It can be a candidate because a mouse with a humanized version of the gene can then be tested for its reaction a the teating drug.

*10.42 One application of DNA fingerprinting technology has been to identify stolen children and return them to their parents. Bobby Larson was taken from a supermarket parking lot in New Jersey in 1978, when he was 4 years old. In 1990, a 16-year-old boy called Ronald Scottwas found in California, living with a couple named Susan and James Scott, who claimed to be his parents. Authorities suspected that Susan and James might be the kidnappers and that Ronald Scott might be Bobby Larson. DNA samples were obtained from Mr. and Mrs. Larson and from Ronald, Susan, and James Scott. Then DNA fingerprinting was done, using a multilocus probe for a particular VNTR family, with the results shown in the following figure. From the information in the figure, what can you say about the parentage of Ronald Scott? Explain. Table

James and Susan Scott are not the parents of "Ronald Scott", because there are several bands in the fingerprint of the boy that are not present in either James or Susan and this could not have been inherited from ether of them.

10.29 What modifications are made to the polymerase chain reaction (PCR) to use this method for site-specific mutagenesis?

Primers that have been modified to incorporate a mutation at a particular site. PCR amplification.

*10.38 Abbreviations used in genomics typically facilitate the quick and easy representation of longer tongue twisting terms. Explore the nuances associated with some abbreviations by stating whether an RFLP, VNTR, or STR could be identified as an SNP? Explain your answers.

SNP can alter the site recognized by a restriction endonuclease, A SNP can also be a RFLP. STRs and VNTRs, repeat sequneces.

10.37 What different types of DNA polymorphisms exist and what different methods can be used to detect them?

SNPs which alter the restriction site and detect fragment length polymorphism. STRs and VNTRs repeat the sequence from a few times to about 100 times.

*10.16 A researcher interested in the control of the cell cycle identifies three different yeast mutants whose rate of cell division is temperature-sensitive. At low, permissive temperatures, the mutant strains grow normally and produce yeast colonies having a normal size. However, at elevated, restrictive temperatures, the mutant strains are unable to divide and produce no colonies. She has a yeast genomic library made in a plasmid E. coli-yeast shuttle vector, and wants to clone the genes affected by the mutants. What steps should she take to accomplish this objective?

She should clone the genes by complementation, transform each mutant with a library containing wild type sequences and the plate the transformants at an elevated restrictive temperature. Purify the plasmid from these colonies, and characterize the cloned gene. Then temperature sensitive phenotypes of the cell division mutants enables the direct selection from transformants receiving the wild type gene.

*10.11 A piece of DNA that is 5,000 bp long is digested with restriction enzymes A and B, singly and together. The DNA fragments produced are separated by DNA electrophoresis and their sizes are calculated, with the following results: Table Each A fragment is extracted from the gel and digested with enzyme B, and each B fragment is extracted from the gel and digested with enzyme A. The sizes of the resulting DNA fragments are determined by gel electrophoresis, with the following results:

Start with the largest fragments. The 1,900 bp fragment produced by digestion with both A and B is part of the 2,100bp fragment produced by digestion with A, and the 2,500 bp fragment produced by digestion with B. Thus the 2,500 bp fragment overlaps by 1,900 bp, leaving a 200-bp A-B fragment on one side and a 600 bp A-B fragment on the other. (SEE SOLUTIONS MANUEL FOR DATA)

10.9 Explain how gel electrophoresis can be used to determine the sizes of the fragments produced by a restriction digest or the size of a PCR product

The DNA sample should be loaded into one well of an agrose gel and a size standard, or marker, DNA sample should be loaded into another well of the gel. After electrophoresis the gel should be stained with a dye, that binds DNA with a fluoresce under ultraviolet light, and the distance each DNA band has migrated from the loading should be measured to migrating distance (in mm).

*10.34 The maps of the sites for restriction enzyme R in the wild type and the mutated cystic fibrosis genes are shown schematically in the following figure: Table here Samples of DNA obtained from a fetus (F) and her parents (M and P) were analyzed by gel electrophoresis followed by the Southern blot technique and hybridization with the radioactively labeled probe designated "CF probe" in the previous figure. The autoradiographic results are shown in the following figure: Figure here Given that cystic fibrosis results from a recessive trait and affected individuals always have two mutant alleles, will the fetus be affected? Explain.

The fetus will only have 1 mutant chromosome.

*10.20 During Southern blot analysis, DNA is separated by size using gel electrophoresis, and then transferred to a membrane filter. Before it is transferred, the gel is soaked in an alkaline solution to denature the doublestranded DNA, and then neutralized. Why is it important to denature the double-stranded DNA? (Hint: Consider how the membrane will be probed.)

The gel is soaked in an alkaline solution to denature the DNA to single stranded form. It must be bound to the membrane in single stranded form so that the probe can bind in a sequence specific manner using complementary bp.

*10.24 Imagine that you have cloned the structural gene for an enzyme that functions in the biosynthesis of catecholamines in the adrenal gland of rats. How could you use this cloned DNA as a probe to determine whether this same gene functions in the rat brain?

The gene should be transcribed into a precursor mRNA, processed to a mature mMRA and the translated to produce the functional enzyme.

*10.28 Katrina purified a clone from a plasmid library made using genomic DNA and sequenced a 500-bp long segment using the dideoxy sequencing method. Her twin-sister Marina used PCR with Taq DNA polymerase to amplify the same 500-bp fragment from genomic DNA. Marina sequenced the fragment using the dideoxy sequencing method, and obtained the same sequence as Katrina did. She then cloned the fragment into a plasmid vector and, following ligation and transformation into E. coli, sequenced several, independently isolated plasmids to verify that she had cloned the correct sequence. Most of them have the same sequence as Katrina's clone, but Marina finds that about 1/3 of them have a sequence that differs in one or two base-pairs. None of the clones that differ from Katrina's clone are identical. Fearing she has done something wrong, Marina repeats her work, only to obtain the same results: about 1/3 of the fragments cloned from the PCR product have single base-pair differences. Explain this discrepancy.

The insert of Katrina sequence was obtained from genomic DNA while the insert of Marina was from PCR. Taq DNA polymerase introduces errors during PCR sot that individual double stranded molecules that are amplified during PCR may have small amounts of variation. If PCR products are sequenced directly a population of molecule is sequences and tha mount of variation is small enough to not be noticed and there for a small molecules may have an error. But when PCR is clones each independently isolated plasmid has an insert derived from a different double stranded DNA PCR product so that the error is apparent.

10.50 The ability to place cloned genes into plants raises the possibility of engineering new, better strains of crops such as wheat, maize, and squash. It is possible to identify useful genes, isolate them by cloning, and insert them directly into a plant host. Usually these genes bring out desired traits that allow the crops in question to flourish. Why then is there such concern by consumers about this process? Do you feel that the concern is justified? Defend your answer.

There is clearly a huge potential for success in crop modification. Your personal opinion.

10.2 Phage vectors used for cloning kill the host bacterial cell in which they are propagated. How can this be advantageous for working with DNA clones? What advantages do phage vectors have over plasmid vectors?

They are advantageous with DNA clones because each plaque contains a large number of identical phage. Phage vectors have advantage over plasmid vectors because phase vectors accept larger insects than do plasmids; and many more plaques can "fit" on a plate than can bacterial colonies. So you can get a larger set of clones and a greater amount of "insert" DNA to be screened.

10.8 Some thermostable DNA polymerases used in PCR leave an unpaired A nucleotide at the ends of the amplified fragments. How can this be useful to clone the PCR products?

To clone the amplification product efficiently.

10.31 Metalloproteases are enzymes that require a metal ion as a cofactor when they cleave peptide bonds. Members of one family of metalloproteases share the following consensus amino acid sequence in their catalytic site: His-Glu-X-Gly-His-Asp-X-Gly-X-X-His-Asp (X is any amino acid). Structural models of the catalytic site developed from X-ray crystallographic data suggest that the second amino acid, glutamate, is essential for proteolytic activity. Outline the experimental steps you would take to test this hypothesis. Assume you possess a cDNA encoding a metalloprotease having the consensus sequence, and can measure metalloprotease activity in a biochemical assay.

Use a site-specific mutagenesis to introduce a single base change into the cDNA to alter the primary structure. Then replace a segment of the wild type cDNA and that of the mutant cDNA and purify the proteins that are produced. Finally measure the metalloprotease activity of each protein in the biochemical assay.

*10.45 Male sexual behavior in Drosophila (fruit fly) is under the control of several regulatory genes, including a gene called fruitless. This gene has been cloned, and both genomic and cDNA clones are available. It encodes proteins that appear to function as male-specific transcriptional regulators. One means to understand more fully the function of fruitless in male sexual behavior is to identify genes for proteins that interact with its protein product. Describe the steps you would take to accomplish this goal.

Use an interaction trap assay. Fuse the coding region of a protein produced by fruitless to the sequence of the Gal4p BD, and contransform this plasmid into yeast with a plasmid library containing the GAL 4p AD sequence, which is fused to protein sequence encode by different cDNAs from the Drosophila brain. Purify colonies that express the reporter gene.

10.17 The amino acid sequence of the actin protein is conserved among eukaryotes. Outline how you would use a genomic library of yeast prepared in a bacterial plasmid vector and a cloned cDNA for human actin to identify the yeast actin gene

Use the cloned cDNA for human actin as a heterologous probe to screen the yeast actin gene. After cell lysis, the plasmid DNA will bind to the filters so that colonies with inserts can be identified using the heterologous probe.

10.49 What methods are used to introduce genes into plant cells, and how are these methods different than those used to introduce genes into animal cells?

Virus infected vectors and Electroporation. To transform monotyledonous plants, electroporation and gene gu methods are used to introduce DNA into plant cells.

10.47 Just as VNTRs and STRs can be used in forensic analyses to determine human genetic identity, they can be used to determine the genetic identity of members of an endangered species. This can be helpful to track animals poached from protected reserves and associate parts of endangered animals that are sold illegally with their source. How would you identify a set of polymorphic STR loci containing a CAG repeat in an endangered species?

You can determine whether any of the loci containing CAG repeats have polymorphic lengths of CAG repeats to design primers for PCR amplification. Isolating DNA from tissue samples taken from multiple members of the population, amplify the different loci containing CAG repeats using PCR and template DNA from the different individuals, and separating the amplification products by size using gel electrophoresis.

*10.12 A colleague has sent you a 4,500-bp DNA fragment excised from a plasmid cloning vector with the enzymes PstI and BglII (see Table 8.1, p. 174, for a description of these enzymes and the sites they recognize). Your colleague tells you that within the fragment there is an EcoRI site that lies 490 bp from the PstI site. a. List the steps you would take to clone the PstI-BglII DNA fragment into the plasmid vector pBluescript II (described in Figure 8.4, p. 176). b. How would you verify that you have cloned the correct fragment and how would you determine its orientation within the pBluescript II cloning vector?

a. (SEE SOLUTIONS MANUEL FOR FURTHER EXPLANATION AND DATA) b. Transform the ligated DNA into a host bacterial cell, and plate the cells on baterial medium containing ampicillin and a substrate for b-galactosidase that turns blue when cleaved.

*10.21 A researcher digests genomic DNA with the restriction enzyme EcoRI, separates it by size on an agarose gel, and transfers the DNA fragments in the gel to a membrane filter using the Southern blot procedure. What result would she expect to see if the source of the DNA and the probe for the blot is described as follows? a. The genomic DNA is from a normal human. The probe is a 2.0-kb DNA fragment excised by the enzyme EcoRI from a plasmid containing single-copy genomic DNA. b. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE ("long interspersed element", a type of repetitive sequence; see Chapter 2, p. 29 and Chapter 7, pp. 160-161) that has an internal EcoRI site. c. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE that lacks an internal EcoRI site. d. The genomic DNA is from a human heterozygous for a translocation (exchange of chromosome parts) between chromosomes 14 and 21. The probe is a 3.0-kb DNA fragment that is obtained by excision with the enzyme EcoRI from a plasmid containing single-copy genomic DNA from a normal chromosome 14. The translocation breakpoint on chromosome 14 lies within the 3.0-kb genomic DNA fragment. e. The genomic DNA is from a normal female. The probe is a 5.0-kb DNA fragment containing part of the testis determining factor TDF gene, a gene located on the Y chromosome.

a. A 2 kb band because it is a single copy genomic DNA sequence. b. LINES are moderately repetitive DNA sequences, which may be distributed throughout the genome. The DNA will be cut into 2 fragments by EcoRI during preparation of the southern blot. When the blot in incubated with the probe, both hybrids will hybridize to the probe. c. Similar to B. The sizes of the bands seen reflect the distances between EcoRI sites that flank a LINE. All of the bands will be larger than the elements because the element is not cleaved by EcoRI. Counting the number of bands can give an estimate of the number of copies of the element in the genome. d. Since chromosome 14 has a break point in the 3 kb EcoRI fragment the 3 kb fragments is now split into 2 parts, each attached to a different segment of chromosome 2. The 3 kb probe spans the translocation break point and will bind to 2 different fragments, one from each translocation chromosomes. e. Since the TDF is on the Y chromosomes, no signal should be seen in a southern blot prepared with DNA from a female having only X chromosomes.

*10.43 As described in the text and demonstrated in Question 10.42, VNTRs can robustly distinguish between different individuals. Five well-chosen, single locus VNTR probes used together can almost uniquely identify one individual because, statistically, they are able to discriminate 1 in 10^9 individuals. However, the use of VNTR markers has largely been supplanted by the use of STR markers. For example, the FBI uses a set of 13 STR markers in forensic analyses. Different fluorescently labeled primers and reaction conditions have been developed so that this marker set can be multiplexed—all of the markers can be amplified in one PCR reaction. The marker set used by the FBI, the number of alleles at each marker, and the probability of obtaining a random match of a marker in Caucasians is listed in the following table: Table Here a. Consider the types of DNA samples that the FBI analyzes and the requirements concerning DNA samples in the methods used to analyze STR and VNTR markers. Why is the use of STR markers preferable to the use of VNTR markers? b. Why is it advantageous to be able to multiplex the PCR reactions used in forensic STR analyses? c. Suppose the first four STR markers listed in the table are used to characterize the genotype of an individual, and the genotype is an exact match with results obtained from a hair sample found at a crime scene. What is the probability that the individual has been misidentified, that is, what is the chance of a random match when just these four markers are used? About how often do you expect an individual to be misidentified if only these four markers are used? d. Answer the questions posed in (c) if all 13 STR markers are used.

a. Because the PCR method requires very small amounts of template DNA, and if the primers are designed to amplify only small regions, even DNA that is degraded partially can be used. b. Multiplexing PCR reactions ensure that the different STR results obtained in the reaction are all derived from a single DNA sample and limited amounts of DNA samples are used efficiently. c. 1 out of 15,702 d. 1 person in 5.94 x 10^14

10.1 Much effort has been spent on developing cloning vectors that replicate in organisms other than E. coli. a. Describe several different reasons one might want to clone DNA in an organism other than E. coli. b. What is a shuttle vector, and why is it used? c. Describe the salient features of a vector that could be used for cloning DNA in yeast

a. Because they are useful for studying eukaryotic genes in their environment, commercial production in eukaryotic gene products (drugs and antibodies), developing gene therapy, engineering crop plants, and developing transgenic animals. b. Shutter vectors are cloning vectors that can replicate in 2 or more host organisms. They are used to introduce DNA to an organism other than E.coli, and has the ability to replicate in E.coli, which is faster and easier. c. It must contain dominant selectable markers for both yeast and E.coli; have several unique restriction enzyme sites for cloning foreign DNA; and have a sequence that allows it to replicate autonomously.

*10.33 DNA was prepared from small samples of white blood cells from a large number of people. These DNAs were individually digested with EcoRI, subjected to electrophoresis and Southern blotting, and the blot was probed with a radioactively labeled cloned human sequence. Ten different patterns were seen among all of the samples. The following figure shows the results seen in ten individuals, each of whom is representative of a different pattern. Table here a. Explain the hybridization patterns seen in the 10 representative individuals in terms of variation in EcoRI sites. b. If the individuals whose DNA samples are in lanes 1 and 6 on the blot were to produce offspring together, what bands would you expect to see in DNA samples from these offspring?

a. Different patterns of hybridizing fragments because of polymorphism of the EcoRI sites in the region. b. 1 is homozygous so it will be present in all of the offspring. 6 will contribute chromosomes of 2 kinds.

10.23 Sara is an undergraduate student who is doing an internship in the research laboratory described in Questions 10.14 and 10.22. Just before Sara started working in the lab, the restriction map in Figure 10.D was made of the 47-kb NotI restriction fragment containing the prion protein gene (distances between restriction sites are in kb). Since smaller DNA fragments cloned into plasmids are more easily analyzed than large DNA fragments cloned Table here into BACs, Sara has been asked to "subclone" the 6.1-, 10.5-, 4.1- and 8.2-kb BamHI DNA fragments containing the prion-protein gene into the pBluescript II plasmid vector (see Figure 8.4, p. 174, for a description of pBluescript II). Her mentor gives her some intact pBluescript II plasmid DNA, some of the purified 47-kb NotI fragment, and shows her where the stocks of DNA ligase, BamHI, and reagents for PCR are stored in the lab. a. Describe the steps Sara should take to complete her task if she has no information about the sequence of the 47-kb NotI fragment. In your answer, address how she will identify plasmids that contain genomic DNA inserts, and how she will verify that she has identified clones containing each of the desired genomic BamHI fragments. b. Describe an alternative approach that Sara could take to complete her task if she first performs a bioinformatic analysis utilizing DNA sequence information available from the mouse genome project, and identifies the sequence of the 47-kb NotI fragment

a. Digest the pBluescript II and 47 bk NotI fragment DNAs with BamHI. The separate the digestion products using gel electrophrosis and purify the genomic DNA fragments. Then set up 4 ligation reactions, one to purify genomic DNA fragments and some of the BamHI digested pBluescript II DNAs. Then separate transform each of the ligations into E.coli and select for bacteria on media. Finally verify that the white colonies from each transformation have the desired genomic DNA inserts. b. PCR amplifications to amplify each of the four desired fragments. And design promers that will amplify a template that spans just beyond an adjacent pair of BamHI sites so that the amplification products can be cleaved with BamHIand the cloned into pBluescript II.

*10.35 The enzyme Tsp45I recognizes the 5-bp site 5' -G-T-(either C or G)-A-C-3' . This site appears in exon 4 of the human gene for -synuclein, where, in a rare form of Parkinson disease, it is altered by a single G-to-A mutation. (Note: Not all forms of Parkinson disease are caused by genetic mutations.) a. Suppose you have primers that can be used in PCR to amplify a 200-bp segment of exon 4 containing the Tsp45I site, and that the Tsp45I site is 80 bp from the right primer. Describe the steps you would take to determine if a patient with Parkinson disease has this alpha-synuclein mutation. b. What different results would you see in homozygotes for the normal allele, homozygotes for the mutant allele, and in heterozygotes? c. How would you determine, in heterozygotes, if the mutant allele is transcribed in a particular tissue?

a. Isolate genomic DNA from the individual with Parkinsons and use the PCR to amplify thee 200 bp segment of exon 4, purify the PCR product, digest it and resolve the digestion by using gel electrophoresis. b. homozygotes for the normal allele would have 120 and 80 bp fragments. Homozygotes for the mutant allele would have 200 bp fragments. Heterozygotes would have 200, 120, and 80 bp fragments. c. By using RT-PCR to amplify a DNA copy of the mRNA, and digest the RT-PCR product with Tsp45I.

*10.19 A 10-kb genomic DNA EcoRI fragment from a newly discovered insect is ligated into the EcoRI site of the pBluescript II plasmid vector (described in Figure 8.4, p. 176) and transformed into E. coli. Plasmid DNA and genomic DNA from the insect are prepared and each DNA sample is digested completely with the restriction enzyme EcoRI. The two digests are loaded into separate wells of an agarose gel, and electrophoresis is used to separate the products by size. a. What will be seen in the lanes of the gel after it is stained to visualize the size-separated DNA molecules? b. What will be seen if the gel is transferred to a membrane to make a Southern blot, and the blot is probed with the 10-kb EcoRI fragment? (Assume the fragment does not contain any repetitive DNA sequence.)

a. It will have a smear that reflects the large number and many different sizes of EcoRI fragments. b. The probe will detect the 10 kb EcoRI fragment specifically, so a signal will be seen in each lane at 10 kb.

*10.22 The investigators described in Question 10.14 were successful in purifying a BAC clone containing the gene for the mouse prion protein. To narrow down which region of the BAC DNA contains the gene for the prion protein gene, they purified the BAC DNA, digested it with the restriction enzyme NotI, and separated the products of the enzymatic digestion by size using gel electrophoresis. Then they purified each of the relatively large NotI DNA fragments from the gel, digested each individually with the restriction enzyme BamHI, and separated the products of each enzymatic digestion by size using gel electrophoresis (see Table 8.1, p. 174, for a description of the sites recognized by NotI and BamHI). Finally, they transferred the size-separated DNA fragments from the agarose gel onto a membrane filter using the Southern blot technique, and allowed the DNA fragments on the filter to hybridize with a labeled cDNA probe. Figure 10.C shows the results that were obtained: The pattern of DNA bands seen after the BAC DNA is digested with NotI is shown in Panel A, the pattern of DNA bands seen after each NotI fragment is digested with BamHI is shown in Panel B, and the pattern of hybridizing DNA fragments visible after probing the Southern blot is shown in Panel C. a. Note the scales (in kb) on the left of each figure. Why are relatively larger DNA fragments obtained with NotI than with BamHI? b. An alternative approach to identify the BamHI fragments containing the prion-protein gene would be to digest the BAC DNA directly with BamHI, separate the products by size using gel electrophoresis, make a Southern blot, and probe it with the labeled cDNA clone. Why might the researchers have added the additional step of first purifying individual large NotI fragments, and then separately digesting each with BamHI before making the Southern blot? c. Which NotI DNA fragment contains the gene for the mouse prion protein? d. Which BamHI fragments contain the gene for the mouse prion protein? e. About what size is the RNA-coding region of the gene for the mouse prion protein? Why is it so much larger than the cDNA?

a. NotI recognizes an 8 bp site while BamHI recognizes a 6 pb site. NotI fragments are larger than BamHI. b. There are many BamHI fragments in the BAC DNA insert and fewer NotI. Digesting 1st with NotI regions of the BAC to be evaluated in an orderly systematic manner and allows for the BamHI fragments containing the gene to be identified more precisely and then purified. c. since the 47 kb fragment is only NotI fragment that has sequences hybridizing to the cDNA it contains a gene. d. The 10.5, 8.2, 6.1 and 4.1 kb BamHI fragments contain the gene since they hybridize to the cDNA probe.

10.13 A researcher has a cDNA for a human gene. a. How should she proceed if she wants to clone the genomic sequence for that gene? b. What kinds of information can be obtained from the analysis of genomic DNA clones that cannot be obtained from the analysis of cDNA clones?

a. She should isolate a fragment of the cDNA inserted into the vector and use the probe to screen a genomic DNA library. b. They provide intro-extron boundaries, transcriptional control regions, and polyadenylation sites, which is important for evaluation hoe a genes expression is controlled.

*10.27 Taq DNA polymerase, which is commonly used for PCR, is a thermostable DNA polymerase that lacks proofreading activity. Other DNA polymerases, such as Vent, have proofreading activity. a. What advantages are there to using a DNA polymerase for PCR that has proofreading activity? b. Although some DNA polymerases are more accurate than others, all DNA polymerases used in PCR introduce errors at a low rate. Why are errors introduced in the first few cycles of a PCR amplification more problematic than errors introduced in the last few cycles of PCR amplification?

a. Significantly reduce the introduction of errors. b. So that fewer molecules have errors.

10.44 About midnight on Saturday, the strangled body of a regular patron of the Seedy Lounge is found in an alleyway near the bar. The police interview the workers and patrons A-R remaining in the bar. A few of the patrons indicate that several individuals, including the bartender, owed money to the victim. The police notice that the bartender and patrons A, C, D, F, K, L, O, and R all have recent cuts and scratches on their faces and backs of their necks, but are told that these happened during mud-wrestling matches earlier in the evening. DNA samples are obtained from the bartender and the bar's patrons, from tissues of the victim, and from scrapings of her fingernails. STR analyses are performed on the DNA samples using three of the markers described in Question 10.43: THO1, D18S51, and D21S11. The sizes of the PCR products obtained in each DNA sample for each marker are shown in Table 10.A. Table a. How many different alleles are present at each marker in these samples and how does this compare to the total number of alleles that exist? How do you explain the appearance of only one marker allele in some individuals? How do you explain the appearance of three and four marker alleles in the DNA sample obtained from the victim's fingernails? b. Who should the police investigate further if they consider the results obtained using only the D21S11 marker? Explain your reasoning. c. Who should the police investigate further if the consider the results obtained using all three STR markers? Explain your reasoning.

a. Since one individual can have at most 2 alleles at 1 locus, the presence of 3 or 4 markers alleles in the DNA sample obtained from the victims fingernails indicates the presence of 2 different DNA samples that of the victim and ta person she had scratched. b. The victim has 221 and 239 pb alleles at the D21S11 locus. c. The victim has the 162 and 170 bp alleles at the THO1 locus.

10.46 Genetic variability is important for maintaining the ability of a species to adapt to different environments. Therefore, it is important to understand how much geneticvariation there is in an endangered species, as this type of information can be used to design better strategies to help the species from becoming extinct. Listed below are four strategies that have been proposed for detecting a SNP in a known DNA sequence in several hundred individuals from an endangered species. Evaluate them critically, and explain why each is, or is not, a good strategy for this purpose. a. Sacrifice each of the animals or plants in the name of science. Isolate their genomic DNA, prepare libraries from each, and screen for clones containing the sequence. Sequence each clone individually. Then compare the sequences of the different clones. b. Isolate a few cells (e.g., by using a cheek scraping or leaf sampling) from each of the individuals. Prepare DNA from the samples and use the ASO hybridization method. c. Isolate a few cells from each of the individuals. Prepare DNA from each of the samples and then use the yeast two-hybrid system. d. Search the literature to find a restriction enzyme that cleaves the sequence containing the SNP and that cleaves the site when only one SNP allele is present. Use the restriction enzyme to measure the site as an RFLP marker. After isolating a few cells from the individuals, prepare DNA from the cells, digest it with the restriction enzyme, separate it by size using electrophoresis, make a Southern blot, and perform an RFLP analysis.

a. The strategy is likely to push the species further toward extinction because it eliminates several hundred individuals and it is labor intensive and costly. b. It is essentially noninvasive and does not harm the organism and it is efficient and cost effective. c. The strategy would not accomplish the objective. d. The SNP may not be able to be assessed using the RFLP method because not all SNPs alter restrictionsites.

10.25 A cDNA library is made with mRNA isolated from liver tissue and the vector shown in Figure 10.4. When a cloned cDNA insert from that library is digested with the enzymes EcoRI (E), HindIII (H), and BamHI (B) (described in Table 8.1, p. 174), the restriction map shown in the following figure, part (a), is obtained. When this cDNA is used to screen a cDNA library made with mRNA from brain tissue and the vector shown in Figure 10.4, three identical cDNAs with the restriction map shown in the following figure, part (b), are obtained. When a uniformly labeled, 32P-labeled riboprobe made using T7 RNA polymerase is prepared using either cDNA and the probe is allowed to hybridize to a Southern blot prepared from genomic DNA digested singly with the enzymes EcoRI, HindIII, and BamHI, an autoradiograph shows the pattern of bands in the following figure, part (c). When any of the -32P labeled riboprobes are used to probe a northern blot prepared with poly(A)+ mRNA isolated from liver and brain tissues, no signal is seen. However, when the same northern blot is probed with a uniformly labeled, -32P labeled probe is prepared using the random primer method (described in Box 10.1), the pattern of bands in part (d) of the figure is seen. Fully analyze these data and then answer the following questions: a. Do these cDNAs derive from the same gene? b. Why are different-sized bands seen on the northern blot? c. Why could hybridization signal be detected on the Southern blot but not on the northern blot when riboprobes were used? Why could hybridization signal be detected on the northern blot when a random-primed probe was used? (Hint: consider how these probes are made and which nucleic acid strands become labeled.) d. Why do the cDNAs have different restriction maps? e. Why are some of the bands seen on the whole genome Southern blot different sizes than some of the restriction fragments in the cDNAs?

a. Yes b. They indicate that the primary mRNA for this gene may be processed differently in brain and liver tissue. c. It can be detected on a southern blot because a single stranded probe will be able to bind to a complementary DNA strand immobilized on the blot. Since there is no seen signal on the northern blot because the riboprobe has the same sequence and polarity as the sense stranded RNA on that blot. d. Because the 2 cDNA are copies of mRNA found in 2 different tissues. e. Because cDNA maps give an indication of the structure of the mRNA transcripts.

10.7 One frequent objective of expressing a protein in E. coli using an expression vector is to purify it. If the expressed protein is "tagged" at one end with a particular peptide sequence, it is easier to purify. For example, proteins tagged at one end with six histidine residues can be recovered from lysed E. coli cells by incubating the lysate with a nickel-containing resin. The six-histidine tag has a high affinity for the resin, facilitating the purification of the protein from the lysate. Proteins tagged in this way are fusion proteins—they contain the amino acid sequence encoded by an open reading frame (ORF) of a cDNA fused to the amino acid sequence of the tag. Some plasmid vectors have been designed to facilitate the production of such fusion proteins. They have an E. coli promoter sequence for transcription initiation and are designed so that the RNA that is produced will have a Shine-Dalgarno sequence near its5' end to facilitate ribosome binding. Following this, they have an ORF with the codons for the tag at its end. A multiple cloning site (MCS) is embedded within the ORF to facilitate cloning of part of a cDNA. Figure 10.B shows the MCS in one such vector. In the figure, the 5'-to-3' DNA strand that has the same polarity as the resulting mRNA is in bold type, the amino acids it encodes are given underneath their codons, and three unique restriction enzyme sites in the vector are shadowed in grey with the sites of DNA cleavage indicated by lines. a. Suppose your want to tag a polypeptide encoded by a cloned cDNA whose ORF includes XhoI and EcoRI sites close to its beginning and a PstI site close to its end. What steps would you take to insert a fragment containing most of the cDNA's ORF into this expression vector? Can you be certain that a fusion protein will be produced? b. How would you clone the ORF of a previously cloned cDNA into this expression vector if the cDNA had no XhoI, PstI, or EcoRI sites? What concerns would you need to address to ensure that a fusion protein would be produced?

a. You would use restriction digestion to cleave the cloned cDNA so that most of the cDNA's ORF is released by the restriction digestion. Then clone the ORF directly into the expression vector. The beginning of ORF must be inserted near the translation start site, and the end of the ORF must be inserted near the 6-histidine tag. The Xhol, EcoRI and PstI sites are located in such a directional cloning can be done only if both the vector and DNA clone are cleaved with XhoI and PstI. Then the Xhol-PstI fragment from the cDNA clone can be inserted into the expression vector in an oreintatuinm that will produce the desired fusion protein. Even if the ORF is inserted in the correct orientation a fusion protein might not be produced. The insert might lead to a frame shift, so that the open reading frame is not maintained within the inserted sequence and the desired fusion protein cannot be produced. b. Then you would have to clone the ORF from the cDNA using PCR-based strategy. To ensure that a fusion protein is produced, carefully design the PCR primers so that the open reading frame is maintained after the digested fragment is ligated into the expression vector.

10.3 What is a cDNA library, and from what cellular material is it derived? How is a cDNA library used in cloning particular genes?

cDNA library is a collection of clones containing the different cDNAs synthesized from the entire population of mRNA of a particular tissue or cell. cDNA library is constructed using mRNA isolated from a particular tissue. cDNA library is used in cloning particular genes by screening for cloned copies of particular mRNA transcripts, either by screening genomic libraries or performing an expression screen.

*10.5 Suppose you wanted to produce human insulin (a peptide hormone) by cloning. Assume that you could do this by inserting the human insulin gene into a bacterialhost where, given the appropriate conditions, the human gene would be transcribed and then translated into human insulin. Which would be better to use as your source of the gene: human genomic insulin DNA or a cDNA copy of this gene? Explain your choice.

cDNA, because it can synthesize from cytoplasmic poly (A) + mRNA does not.