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Question: Describe the molecular features of a BAC


Describe the molecular features of a BAC cloning vector. What is the primary advantage of a BAC vector over a plasmid or viral vector?



> What is the purpose of using a secondary antibody?

> How is the sgRNA different from certain components of the bacterial defense system described in Chapter 17?

> Describe three possible uses of site-directed mutagenesis.

> What needs to happen so the reporter molecule can emit fluorescence that is not quenched?

> After four cycles of PCR, which type of PCR product predominates? Explain why.

> What is horizontal gene transfer?

> What is an advantage of making a cDNA library rather than a genomic library?

> Explain the meaning of the name reverse transcriptase.

> Explain the role of the gene that is the selectable marker gene in this experiment.

> Prior to the action of DNA ligase, how many hydrogen bonds are holding these two DNA fragments together?

> What is the purpose of the rat liver extract in this procedure?

> In people, what is a common cause of thymine dimer formation and in what cell type(s) would it be most likely to occur?

> Does 5-bromouracil cause a transition or a transversion?

> Explain how tandem mass spectroscopy is used to determine the sequence of a peptide. Once a peptide sequence is known, how is this information used to determine the sequence of the entire protein?

> Can two-dimensional gel electrophoresis be used as a purification technique? Explain.

> In the procedure called RNA sequencing (RNA-Seq), what type of molecule is actually sequenced?

> Refer to question 3 in More Genetic TIPS before answering this question. Based on the multiple-sequence alignment in Figure 24.10, what is/are the most probable time(s) that mutations occurred in the human globin gene family to produce the following amin

> Take a look at the multiple-sequence alignment in Figure 24.10 of the globin polypeptides, focusing on amino acids 101 to 148. A. Which of these amino acids are likely to be most important for globin structure and function? Explain why. B. Which are li

> What is the function of reverse transcriptase?

> The goal of many computer programs is to identify sequence elements within a long segment of DNA. What is a sequence element? Give two examples. How is the specific sequence of a sequence element determined? In other words, is it determined by the comput

> To identify the following types of genetic occurrences, would a computer program use sequence recognition, pattern recognition, or both? A. Whether a segment of Drosophila DNA contains a P element (which is a specific type of transposable element) B. W

> In this chapter, we considered a computer program that translates a DNA sequence into a polypeptide sequence. Instead of running this program, a researcher could simply look the codons up in a genetic code table and determine the sequence by hand. What a

> With regard to DNA microarrays, answer the following questions: A. What is attached to the slide? Be specific about the number of spots, the lengths of DNA fragments, and the origin of the DNA fragments. B. What is hybridized to the microarray? C. How

> Explain how DNA microarrays are used in molecular profiling of cancerous tumors.

> The codon change (Gly-12 to Val-12) in human rasH that converts it to oncogenic rasH has been associated with many types of cancers. For this reason, researchers would like to develop drugs to inhibit oncogenic rasH. Based on your understanding of the Ra

> Discuss ways to distinguish whether a particular form of cancer involves an inherited predisposition or is due strictly to (postzygotic) somatic mutations. In your answer, consider that only one mutation may be inherited, but the cancer might develop onl

> An experimental assay for the blood-clotting protein called factor IX is available. A blood sample was obtained from each individual in the following pedigree. The amount of factor IX protein, shown within each symbol on the pedigree, is expressed as a p

> Chapter 21 describes a method known as Western blotting that can be used to detect a polypeptide that is translated from a particular mRNA. In this method, a particular polypeptide or protein is detected by an antibody that specifically recognizes a segm

> A particular disease is found in a group of South American Indians. During the 1920s, many of these people migrated to Central America. In the Central American group, the disease is never found. Discuss whether or not you think the disease has a genetic

> Which of these mechanisms causes the TE to increase in number?

> What is meant by the term genetic testing? How do testing at the protein level and testing at the DNA level differ? Describe five different techniques used in genetic testing.

> Section 25.1 discussed the types of experimental observations that suggest a disease is inherited. Which of these observations do you find the least convincing? Which do you find the most convincing? Explain your answer.

> Which of the following experimental observations suggest that a disease has a genetic basis? A. The frequency of the disease is less likely in relatives that live apart compared with relatives that live together. B. The frequency of the disease is unus

> Contigs are often made using BAC or cosmid vectors. What are the advantages and disadvantages of these two types of vectors? Which type of contig would you make first, a BAC or cosmid contig? Explain.

> What is a contig? Explain how you would determine that two clones in a contig are overlapping.

> A researcher is interested in a gene found on human chromosome 21. Describe the expected results of a FISH experiment using a probe that is complementary to this gene. How many spots would you see if the probe was used on a sample from an individual with

> Explain how DNA probes with different fluorescence emission wavelengths can be used in a single FISH experiment to map the locations of two or more genes. This method is called chromosome painting. Explain why this is an appropriate term.

> Figure 23.2 describes the technique of FISH. Why is it necessary to fix the cells (and the chromosomes inside of them) to the slides? What does it mean to fix them? Why is it necessary to denature the chromosomal DNA? From Figure 23.2: Sister chrom

> The cells from a person’s malignant tumor were subjected to in situ hybridization using a probe that recognizes a unique sequence on chromosome 14. The probe was detected only once in each of the cells. Explain this result, and speculate on its significa

> Describe the technique of in situ hybridization. Explain how it can be used to map genes.

> Explain what happened to the b allele that allowed gene conversion to occur.

> Discuss where protists are found in this newer organization of eukaryotic species.

> In an in situ hybridization experiment, what is the relationship between the base sequence of the probe DNA and the site on the chromosomal DNA where the probe binds?

> What is an STS? How are STSs generated experimentally? What are the uses of STSs? Explain how a microsatellite can be a polymorphic STS.

> Place the following stages of a physical mapping study in their most logical order: A. Clone large fragments of DNA to make a BAC library. B. Determine the DNA sequence of subclones from a cosmid library. C. Subclone BAC fragments to make a cosmid lib

> Take a look at question 3 in More Genetic TIPS. Let’s suppose a male is heterozygous for two polymorphic sequence-tagged sites. STS-1 exists in two sizes: 211 bp and 289 bp. STS-2 also exists in two sizes: 115 bp and 422 bp. A sample of

> In the Human Genome Project, researchers have collected linkage data from many crosses in which the male was heterozygous for molecular markers and many crosses where the female was heterozygous for the markers. The distance between the same two markers,

> An experimenter used primers that recognize nine different STSs to test their presence in five different BACs. The results are shown here. Draw a contig that maps the alignment of the five BACs. Alignment of STSS and BACS STSS 1 2 3 4 5 7 8 9 6 BACS

> A woman has had five children with two different men. This group of seven individuals is analyzed with regard to three different STSs: STS-1 is 146 bp and 122 bp; STS-2 is 102 bp and 88 bp; and STS-3 is 188 bp and 204 bp. The mother is homozygous for all

> Is each of the following a method used in linkage, cytogenetic, or physical mapping? A. Fluorescence in situ hybridization (FISH) B. Conducting two-factor crosses to compute map distances C. Chromosome walking D. Examination of polytene chromosomes i

> What is molecular pharming? Compared with the production of proteins by bacteria, why might it be advantageous?

> Describe the structure and location of a D-loop.

> Evidence [see P. G. Shiels, A. J. Kind, K. H. Campbell, et al. (1999), “Analysis of telomere lengths in cloned sheep,” Nature 399, 316– 317] suggests that Dolly may have been genetically older than her actual age. As mammals age, the chromosomes in somat

> In the study of plants and animals, it is relatively common for researchers to identify a gene using molecular techniques without knowing the function of the gene. In the case of mice, the function of the gene can be investigated by making a gene knockou

> What is a gene knockout? Is an animal or plant with a gene knockout a heterozygote or homozygote? What might you conclude if a gene knockout does not have a phenotypic effect?

> List and briefly describe five methods for the introduction of cloned genes into plants.

> To produce transgenic plants, plant tissue is exposed to Agrobacterium tumefaciens and then grown in media containing kanamycin, carbenicillin, and plant growth hormones. Explain the purpose behind each of these three agents. What would happen if you lef

> In the procedure in Figure 22.1, why was it necessary to link the coding sequence for the A or B chains to the sequence for β-galactosidase? How were the A or B chains separated from β-galactosidase after the fusion pr

> In the Western blot shown here, proteins were isolated from red blood cells and muscle cells from two different individuals. One individual was unaffected, and the other suffered from a disease known as thalassemia, which involves a defect in hemoglobin.

> The method of Northern blotting is used to determine the amount and size of a particular RNA transcribed in a given cell type. Alternative splicing (discussed in Chapter 12) produces mRNAs of different lengths from the same gene. The Northern blot shown

> Let’s suppose an X-linked gene in mice exists as two alleles, which we will call B and b. X-chromosome inactivation, a process in which one X chromosome is turned off, occurs in the somatic cells of female mammals (see Chapter 5). Allele B encodes an mRN

> What is the purpose of a Northern blotting experiment? What types of information can it tell you about the transcription of a gene?

> Explain why a heteroduplex region may be produced after branch migration occurs.

> Bacillus thuringiensis makes toxins that kill insects. These toxins must be applied several times during the growth season to prevent insect damage. As an alternative to repeated applications, one strategy is to apply bacteria directly to leaves. However

> In Northern and Western blotting, what is the purpose of gel electrophoresis?

> Northern blotting depends on the phenomenon of the binding of a probe to mRNA. In this technique, explain why binding occurs.

> Gene mutagenesis is also used to explore the structure and function of proteins. For example, changes can be made to the coding sequence of a gene to determine how alterations in the amino acid sequence affect the function of a protein. Letâ€&

> Let’s suppose you want to use site-directed mutagenesis to investigate a DNA sequence that functions as a response element for hormone binding. From previous work, you have narrowed down the response element to a sequence of DNA that is 20 bp in length w

> A portion of the coding sequence of a cloned gene is shown here: 5΄–GCCCCCGATCTACATCATTACGGCGAT–3΄ 3΄–CGGGGGCTAGATGTAGTAATGCCGCTA–5΄ This portion of the gene encodes a polypeptide with the amino acid sequence alanine–proline–aspartic acid–leucine–histid

> A sample of DNA was subjected to automated DNA sequencing and the output is shown here. What is the sequence of this DNA segment? T= Red C- Blue G- Black A- Green

> Several research studies are under way that involve the use of gene therapies to inhibit the growth of cancer cells. As discussed in Chapter 25, oncogenes are mutant genes that are overexpressed and cause cancer. New gene therapies are aimed at silencing

> Treatment of adenosine deaminase (ADA) deficiency is an example of ex vivo gene therapy. Why is this therapy called ex vivo? Can ex vivo gene therapy be used to treat all inherited diseases? Explain.

> Researchers have identified a gene in humans that (when mutant) causes severe dwarfism and mental impairment. This disorder is inherited in an autosomal recessive manner, and the mutant allele is known to be a loss-of-function mutation. The same gene has

> Which of these repair systems is particularly valuable to plants?

> What is reproductive cloning? Are identical twins in humans considered to be clones? With regard to agricultural species, what are some potential advantages to reproductive cloning?

> Recombinant bacteria can produce hormones that are normally produced in humans. Briefly describe how this is accomplished.

> Why is a thermostable form of DNA polymerase (e.g., Taq polymerase) used in PCR? Is it necessary to use a thermostable form of DNA polymerase in the dideoxy method or in sitedirected mutagenesis?

> Some vectors used in cloning experiments contain bacterial promoters that are adjacent to unique cloning sites. This makes it possible to insert a gene sequence next to the bacterial promoter and express the gene in bacterial cells. These vectors are cal

> What is a DNA library? Do you think this name is appropriate?

> What is a recombinant vector? How is a recombinant vector constructed? Explain how X-Gal is used in a method of identifying recombinant vectors that contain segments of chromosomal DNA.

> In your own words, describe the series of steps necessary to clone a gene.

> How does gene cloning produce many copies of a gene?

> Describe the important features of cloning vectors. Explain the purpose of selectable markers in cloning experiments.

> Table 21.3 describes the cleavage sites of five different restriction enzymes. After these restriction enzymes have cleaved the DNA, four of them produce sticky ends that can hydrogen bond with complementary sticky ends, as shown in Figure 21.1. The effi

> What is the advantage of genetic recombination, which is depicted in part (b)? From Figure 20.1: DO DO A da Two with Meiosis is parental genotype A crossover occurs between homologous chromatids. completed to yield 4 haploid cells. A B b Two with re

> DNA sequencing can help us to identify mutations within genes. The following data are derived from an experiment in which a normal gene and a mutant gene have been sequenced: Locate and describe the mutation G= Yellow A- Green T- Red C-

> What phase of PCR (exponential, linear, or stationary) is analyzed to quantitate the amount of DNA or RNA in a sample? Explain why this phase is chosen.

> What type of probe is used for real-time PCR? Explain how the level of fluorescence correlates with the level of PCR product.

> Starting with a sample of RNA that contains the mRNA for the β-globin gene, explain how you could create many copies of the β-globin cDNA using reverse transcriptase PCR.

> What is the functional significance of sticky ends in a cloning experiment? What type of bonding makes the ends sticky?

> A bacterium is exposed to a drug that inhibits the N protein. What would you expect to happen if the bacterium was later infected by phage λ? Would phage λ follow the lytic cycle, the lysogenic cycle, or neither? Explain your answer.

> Experimentally, when an E. coli bacterium already has a λ prophage integrated into its chromosome, another λ phage cannot usually infect the cell and establish the lysogenic or lytic cycle. Based on your understanding of the genetic regulation of the pha

> A researcher identified a mutation in PR of phage λ that causes its transcription rate to be increased 10-fold. Do you think this mutation would favor the lytic or lysogenic cycle? Explain your answer.

> Richard Boyce and Paul Howard-Flanders conducted an experiment that provided biochemical evidence that thymine dimers are removed from DNA by a DNA repair system. In their studies, bacterial DNA was radiolabeled so the amount of radioactivity reflected t

> Gerald Rubin and Allan Spradling devised a method of introducing a transposon into Drosophila. This approach has been important for the transposon tagging of many Drosophila genes. The researchers began with a P element that had been cloned on a plasmid.

> What is a reactive oxygen species?

> Tumor-suppressor genes are normal human genes that prevent uncontrollable cell growth. Starting with a normal laboratory human cell line, describe how you could use transposon tagging to identify tumor-suppressor genes. (Note: When a TE hops into a tumor

> Compare and contrast anti-miRNA oligonucleotides, locked nucleic acids (LNAs), and antagomirs, which may eventually be used to treat certain forms of cancer. 

> As described in Chapter 21, the CRISPR-Cas system has been modified so it can be used as a gene mutagenesis tool (look ahead to Figure 21.13). Describe how the gene mutagenesis tool works, and explain how the natural CRISPR-Cas system is altered to produ

> Explain how the data of Fire and Mello suggested that doublestranded RNA is responsible for the silencing of mex-3 mRNA.

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