iGenetics: A Molecular Approach, 3e (Russell/Bose)
Chapter 8 Genomics: The Mapping and Sequencing of Genomes
MATCHING
Please select the best match for each term.
A) Eukaryotic cloning vector
B) Molecule used in DNA sequencing
C) Origin of replication
D) Small circular fragment of DNA
E) Endonuclease isolated from bacteria
1) Restriction enzyme
Skill:Factual recall
2)ARS
Skill: Factual recall
3) Plasmid
Skill: Factual recall
4) YAC
Skill: Factual recall
5) Dideoxynucleotides
Skill: Factual recall
Answers: 1) E 2) C 3) D 4) A 5) B
MULTIPLE CHOICE
6) When DNA is cut with a restriction enzyme, the resulting fragments have
A) 5' carboxyls.
B) 5' hydroxyls.
C) 3' hydroxyls.
D) 3' phosphates.
E) 3' carboxyls.
Answer: C
Skill: Factual recall
7) A plasmid used as a cloning vector in E. coli must have
A) an ori sequence.
B) a selectable marker.
C) unique restriction sites.
D) B and C
E) All of these
Answer: E
Skill: Factual recall
8) Bacteria protect their own DNA from restriction enzyme damage by adding ______groups to certain nucleotides.
A) methyl
B) hydroxyl
C) phosphate
D) amino
E) carboxyl
Answer: A
Skill: Factual recall
9) A polylinker region in a cloning plasmid is characterized by
A) multiple methyl groups.
B) multiple restriction cut sites.
C) multiple reporter genes.
D) multiple expression vectors.
E) multiple cloning targets.
Answer: B
Skill: Factual recall
10) In a physical map, distances are measured in
A) recombination frequencies.
B) number of SNPs.
C) base pairs.
D) probabilities.
E) microns.
Answer: C
Skill: Factual recall
11) In DNA electrophoresis, fragment separation is based on
A) fragment size.
B) fragment nucleotide content.
C) fragment sequence.
D) fragment charge.
E) All of these
Answer: A
Skill: Factual recall
12) The chemical "label" that permits visualization of DNA fragments in an automated sequencing machine is
A) 32P.
B) 35S.
C) fluorescent dyes.
D) tritium.
E) None of these
Answer: C
Skill: Factual recall
13) A DNA copy of an mRNA molecule is called
A) dDNA.
B) rDNA.
C) mDNA.
D) shDNA.
E) cDNA.
Answer: E
Skill: Factual recall
14) The first complete nonviral genome sequenced was that of
A) Escherichia coli.
B) Methanococcus.
C) Saccharomyces cerevisiae.
D) the human mitochondrion.
E) Mus musculus.
Answer: D
Skill: Factual recall
15) Many bacteria are naturally capable of taking up endogenous fragments of DNA in their environment, a phenomenon termed
A) conversion.
B) transformation.
C) hybridization.
D) transfection.
E) conjugation.
Answer: B
Skill: Factual recall
16) An SNP always occurs due to
A) a one base-pair change.
B) a three base-pair change.
C) a single restriction site change.
D) a single amino acid change.
E) None of these
Answer: A
Skill: Factual recall
17) The recognition sequences for many restriction enzymes are
A) terminal repeats.
B) tandem repeats.
C) palindromic.
D) inverted repeats.
E) dual repeats.
Answer: C
Skill: Factual recall
18) In addition to restriction enzymes, which of the following enzymes is required to insert a fragment of DNA into a cloning vector?
A) DNA polymerase
B) DNA ligase
C) RNA polymerase
D) Reverse transcriptase
E) Topoisomerase
Answer: B
Skill: Factual recall
19) Which restriction enzyme leaves blunt ends?
A) XbaI
B) SmaI
C) EcoR1
D) BamHI
E) All of these
Answer: B
Skill: Factual recall
20) A restriction enzyme cuts DNA and leaves the following end: CTGCA
G
Which of the following could be the sequence of the corresponding end of the other fragment generated by the enzyme?
A) G
CTGCA
B) C
GTGCA
C) CCGAT
G
D) GGCTA
C
E) None of these
Answer: A
Skill: Problem-solving
21) The ______method was used by the Celera Genomics Corporation to sequence the human genome.
A) FISH
B) whole-genome shotgun
C) clone contig
D) radiation hybrid
E) pedigree analysis
Answer: B
Skill: Factual recall
22) Candidate open reading frames of a genome are identified by searching for
A) a start codon "in frame" with a stop codon.
B) a start codon.
C) introns and exons.
D) a promoter.
E) All of these
Answer: A
Skill: Factual recall
23) Successful insertion of a DNA fragment into the polylinker region of pBluescript II is detected by
A) production of the enzyme β-galactosidase.
B) absence of the enzyme β-galactosidase.
C) the increased size of the plasmid.
D) autoradiography.
E) None of these
Answer: B
Skill: Factual recall
24) In gel electrophoresis, which of the following DNA fragments would travel the farthest distance from the sample well?
A) ATCCCGAT
B) ATCCCG
C) ATCC
D) AT
E) ATCCCGATTGCACGTT
Answer: D
Skill: Conceptual understanding
25) Which types of cloning vector occur in nature?
A) Plasmids and cosmids
B) Plasmids and bacteriophages
C) Plasmids and BACs
D) YACs and BACs
E) Bacteriophages and cosmids
Answer: B
Skill: Factual recall
TRUE/FALSE
26) A DNA copy of a messenger RNA molecule is called complementary DNA or cDNA.
Answer: TRUE
Skill: Factual recall
27) The human genome consists mostly of noncoding DNA.
Answer: TRUE
Skill: Factual recall
28) Cloning vectors are used to introduce novel DNA into cells.
Answer: TRUE
Skill: Factual recall
29) DNA molecules, regardless of size, have a net positive charge.
Answer: FALSE
Explanation: DNA is a negatively charged molecule.
Skill: Factual recall
30) In partial restriction digestion, all available restriction sites will be cut.
Answer: FALSE
Explanation: Partial digestion leaves some sites untouched by chance.
Skill: Factual recall
31) The quality of an assembled sequence does not depend on the sequence coverage.
Answer: FALSE
Explanation: The higher the coverage of the genome, the better the quality of the assembled sequence.
Skill: Conceptual understanding
32) Cloning vectors generally possess unique restriction sites and dominant selectable markers.
Answer: TRUE
Skill: Factual recall
33) A linear strand of DNA with two restriction enzyme cut sites will yield three fragments on digestion.
Answer: TRUE
Skill: Problem-solving
34) A circular DNA molecule with two restriction enzyme cut sites will yield three fragments on digestion.
Answer: FALSE
Explanation: A circular molecule will yield two DNA fragments when cleaved in two places.
Skill: Problem-solving
35) Archaea are typically found in much the same habitats as bacteria.
Answer: FALSE
Skill: Factual recall
SHORT ANSWER
36) Briefly contrast the dideoxy DNA sequencing developed by Fred Sanger with pyrosequencing methods.
Answer: Dideoxy DNA sequencing involves use of altered (dideoxy) nucleotides that cause termination of replication at each site where such a nucleotide is inserted. The pyrosequencing method does not require chain termination. All four deoxynucleotide precursors are present at all times in the dideoxy sequencing reaction, whereas, only one is present at a time in the pyrosequencing reaction. The DNA template is immobilized on a bead in pyrosequencing, and the method of detection is the production of light by utilizing the pyrophosphate produced after the incorporation of a nucleotide. In the dideoxy method, the DNA template is not immobilized and the sequence is read with the help of fluorescent dyes attached to the dideoxy nucleotide molecules.
Skill: Conceptual understanding
37) Describe the basic approach to shotgun genome sequencing.
Answer: Shotgun sequencing involves cutting the genome into multiple, partially overlapping, fragments, each of which is cloned and sequenced. A computer algorithm is used to assemble the complete sequence by identifying contiguous fragments by their overlapping regions.
Skill: Factual recall
38) The restriction enzyme SmaI cuts DNA at the 6-base recognition sequence 5'-CCCGGG-3'. Calculate the frequency of occurrence of this sequence. (Assume a 50% GC content.)
Answer: Assuming a 1:1 AT:CG ratio in the genome and a random distribution of nucleotides, each nucleotide pair will occur with equal frequency (). The 6-nucleotide sequence would then occur with a frequency of ()6, 0.00024, cutting once in every 4,096 base pairs.
Skill: Problem-solving
39) Describe a DNA microarray setup for assaying mutations associated with a hypothetical genetic disease.
Answer: Normal and mutant gene sequences associated with the disease could be affixed to the array in known positions. DNA from a patient and a known nonmutant individual could be separately isolated and labeled (or mRNA could be used, making cDNA for labeling with reverse transcription). Both are applied to the microarray, and whichever labeled fragments that bind with the DNA fragments on the array will colorimetrically reveal which alleles the patient possesses.
Skill: Conceptual understanding
The following scenario applies to the questions below.
A pBluescript II cloning vector is used to introduce a gene of interest into a bacterial strain. The vector is lacking ampR, however.
40) What problem does this result in?
Answer: ampR is the gene conferring resistance to the antibiotic ampicillin. Ampicillin is used to select for bacterial colonies that have been successfully cloned. Without ampR, both successfully cloned and unsuccessfully cloned bacterial colonies will fail to grow if the selection medium contains ampicillin.
Skill: Conceptual understanding
41) Can you think of an alternative means of identifying successful bacterial transformants in this experiment?
Answer: Successful insertion of the gene of interest into the polylinker region will disrupt the lacZ+ gene; such colonies cannot manufacture β-galactosidase and will therefore remain white in the presence of X-gal. Colonies can thus be screened using X-gal. Of course, the bacteria would have to be grown on media without ampicillin, allowing all the colonies to grow.
Skill: Conceptual understanding
42) Exactly why, in dideoxy DNA sequencing, do dideoxynucleotides halt the replication process?
Answer: Dideoxynucleotides are missing the 3' hydroxyl group necessary for linkage with the 5' phosphate group of new nucleotides by DNA polymerase.
Skill: Conceptual understanding
43) What is a hapmap?
Answer: A hapmap is a complete description of all the haplotypes known in a population as well as the chromosomal location of each haplotype.
Skill: Factual recall
44) How can the recircularization of a restriction-digested vector during a ligation reaction be minimized?
Answer: The restriction-digested vector can be treated with alkaline phosphatase to remove the 5' phosphates without which the DNA ligase is unable to form a phosphodiester bond.
Skill: Factual recall
45) What advantage do cDNA libraries have over genomic libraries?
Answer: Eukaryotic genomes tend to have more noncoding regions. Therefore, cDNA libraries offer a way to eliminate the nontranscribed regions and to look at only the transcribed regions of the genome. cDNA libraries can also be made from specific tissues.
Skill: Conceptual understanding
46) Based on DNA sequences of single genes, genomic analysis has confirmed that prokaryotes are divided into two great lineages: the Bacteria and the Archaea. The latter may be more closely related to the Eukarya. Recount the lines of evidence that support these findings.
Answer: Sequence divergence between archaeans and bacteria is considerable, and despite the general morphological similarity of these groups, the Archaea more closely resemble the Eukarya in terms of both the details of DNA replication and the expression (and, in some cases, the structure) of their genes.
Skill: Factual recall
47) Discuss the relative roles of descriptive and hypothesis-driven science in genomics.
Answer: Although sequencing genomes is a descriptive task, the data so obtained provide the raw material for framing an extensive array of hypotheses about gene and genome function, integration, and evolution. This is a good example of how descriptive knowledge is a necessary first step in understanding biological systems.
Skill: Conceptual understanding
48) Sequencing the dog genome is currently a high priority for genomics researchers. What insights do you think can be gained about the evolutionary process by analyzing the genome of dogs?
Answer: Dogs are diversified into a multitude of breeds, many with striking morphological and behavioral differences. Yet, all dogs are the same species, and their DNA differs only minutely. Comparison of the genomes of different dog breeds may therefore shed light on which genetic elements are responsible for the radically different developmental pathways of different dog breeds–a process that may represent a microcosm of species-level differentiation over evolutionary time.
Skill: Conceptual understanding
49) It is not always easy to locate or identify genes based on cDNA. How is this problem created by the differences between cDNA and DNA with respect to a given gene?
Answer: cDNA is made from mRNA, which in eukaryotes is processed transcript with exons removed. The original gene may have many exons, and if so, the cDNA may little resemble it.
Skill: Conceptual understanding
50) Given the DNA oligonucleotide 5'-AGTCTAGGCT-3', reconstruct a sequencing gel based on dideoxy sequencing, showing the relative position of each DNA fragment (band) on the gel.
Answer: The gel drawn by the student should resemble that in Figure 8.11.
Skill: Conceptual understanding
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