Genetics and Biotechnology

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Reading Assignments and Questions: 2011-2012


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Genetics and Biotechnology
Reading Assignments and Questions
Recombinant DNA (3d Ed.)


Unit 1: From Mendel’s Garden to the Double Helix
(Genetics to the Beginning of Genomics)

Text: Ch. 1 (DNA Is the Primary Genetic Material), pp. 3-22.

RA1: pp. 3-4 (Beginning of 3 to End of 4)

RQ1: What is an allele?

RQ2: Draw Punnett Squares to show how Mendel’s monohybrid crosses of heterozygous plants
generated a 3:1 phenotypic ratio and how his dihybrid crosses of heterozygous plants generated a
9:3:3:1 phenotypic ratio. [This is not covered in the text.]

RQ3: What is the difference between segregation (Mendel’s first law) and independent assortment
(Mendel’s second law)? [This is not covered in the text.]

RA2: pp. 5-7 (Discrete Factors of Inheritance…Genes Are Mapped)

RQ4: Distinguish between genotype and phenotype.

RQ5 (Figure 1-1): Review mitosis and meiosis, paying particular attention to distinguishing
metaphase-anaphase of mitosis from metaphase-anaphase of meiosis I.

RQ6: Describe the parallels between the behavior of chromosomes and that expected of Mendel’s
factors.

RA3: pp. 7-9 (Genes Are Mapped…Not All Genes)

RQ7: Explain how Nettie Stevens and Edmund Wilson were able to be the first scientists to assign a
given trait to a particular chromosome. What was the trait? Use a Punnett Square to explain your
answer.

RQ8: Explain how Thomas Hunt Morgan was able to assign a second trait to a particular
chromosome. What was the trait? Use a Punnett Square to explain your answer.

RQ9: What is meant by the term linkage? How can crossing-over disrupt linkage?

RQ10: Why is it more likely that linkage between two genes separated by a large distance on a
given chromosome will be disrupted by crossing-over as compared to two genes separated by a
small distance?

Reading Assignments and Questions: 2011-2012


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RQ11: Define a map unit (centiMorgan).

RQ12 (Figure 1-6): What do you think the symbol “+” represents in the traits for red eye (pr
+
) and
normal wing (vg
+
)? Study the diagram to explain the system for the nomenclature (i.e., the use of
symbols for traits) used here.

RQ13 (Figure 1-6): What general observation can you make as to the relative number of
recombinants and nonrecombinants? Why does this make sense?

RQ14 (Figure 1-6): The flies pictured at the top of the diagram would belong to the F1 generation
and those at the bottom of the diagram would belong to the F2 generation. The parents of the F1
generation would belong to the P generation. Assume that the parents of the wild (phenotypically
normal) F1 fly on the left were both homozygous, one being pr
+
vg
+
/pr
+
vg
+
and the other being
pr

vg/pr

vg. Assume that both parents of the purple-eyed, vestigial-winged F1 fly on the right were
homozygous for the mutant traits, pr vg/pr vg. Show the haploid genotypes of the gametes produced
by the P flies to generate their respective F1 offspring. This represents the gametic input of the P
generation.

RQ15 (Figure 1-6): The gametic output of the F1 generation is represented by the chromosomes
immediately above the F2 flies. In analyzing the gametic output of the wild F1 fly, why is it useful
that the gametic output of the mutant fly is homozygous recessive?

RQ16 (Figure 1-6): Compare the gametic input to and output from the wild F1 fly with the
genotypes of the F2 recombinants. This relationship is the basis for the important definition of
recombination frequency. Generalize from this relationship to define recombination frequency.

RQ17 (Figure 1-6): Why will the recombination frequency for linked traits always be less than
50%? Use a diagram of crossing-over to help explain your answer.

RQ18 (Figure 1-6): The genes for the traits in this diagram are linked, i.e., they are on the same
chromosome. Assume that the traits for purple eyes and vestigial wings were on different
chromosomes, i.e., assume that the traits were unlinked. In this case the parents of the wild F1 fly
would be represented as pr
+
/pr
+
; vg
+
/vg
+
and as pr/pr ; vg/vg and those of the mutant F1 fly would
be represented as pr/pr ; vg/vg. The diploid genotype of the F1 fly would be pr
+
/pr

; vg
+
/vg. Note
the difference in nomenclature for the linked traits described in RQ14 and the unlinked traits
described here. Use a branching diagram to show the genotypes of the four different gametes that
the wild F1 fly could produce. What would the expected percentage be for each of the four gametes
produced by the wild F1 fly? Continuing with the assumption that these traits are not linked, use a
Punnett Square to show the predicted phenotypic ratio of the offspring from a cross between the F1
flies.

RQ19 (Figure 1-6): Referring to the previous question which assumes no linkage, compare the
genetic input to the wild F1 fly with its genetic output. The frequency by which the gametic input
differs from the gametic output is defined as the recombination frequency for these two traits
Recombination frequencies are used regardless of whether or not the traits are linked. What is
the predicted recombination frequency in the case of these unlinked traits? Compare the
Reading Assignments and Questions: 2011-2012


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recombination frequency generated by independent assortment in the case of unlinked traits with the
recombination frequency generated in the case of linkage. What generalization can you make?


RA4: pp. 9-11 (Not All Genes….End of p. 11)

RQ20 (Figure 1-7): In the plant Mirabilis jalapa, what explains the fact the color of the plant is
controlled by the flower that produces the egg rather than the flower that produces the pollen (i.e.,
the sperm)?

RQ21: Why is mitochondrial DNA inherited exclusively from the mother?

RQ22: What discovery by Herman Muller greatly facilitated the study of mutations and also cast a
warning to humans?

RQ23: Why are mutations generally harmful?

RA5: 12-15 (Chromosomes Contain…A Biological Assay)

RQ24: What is the basic structural difference between purines and pyrimidines. Which bases
belong to each group?

RQ25: What was the basic technique used beginning in the 19
th
century to determine the constituent
parts of nucleic acids? Why do you think that this technique did not provide information about the
molecular structure of DNA?

RQ26: If nucleic acid had been a uniform polymer in which each base was repeated every four
nucleotides along the entire polynucleotide chain, as Phoebus Levene believed to be the case, what
would the significance of that fact have been?

RQ27: What did Chargaff’s base analysis establish? Did he draw any clues from this data as to the
structure of DNA?

RQ28: Why did the fact that the same amount of DNA was found in all diploid cells of a given
species whereas half that amount was found in sperm and eggs support the notion that genes could
be made of DNA?

RA6: pp. 15-18(A Biological Assay…DNA Has a Regular)

RQ29 (Figure 1-11): Describe Oswald Avery’s experiment whereby he identified DNA as the
“transforming factor” first identified by Frederick Griffith.

RQ30: What is a bacteriophage (phage)?

RQ31 (Figure 1-12): How did the Hershey-Chase experiment support Avery’s conclusion that DNA
was the “transforming factor”?

Reading Assignments and Questions: 2011-2012


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RA7: pp. 18-22 (DNA Has a Regular…DNA Replication is Semi-Conservative)

RQ32: Why did the A form structure of DNA (Fig. 1-13a), support the theory that DNA had a
precise structure in which the individual DNA molecules could be assumed to have a very similar
form? Who took the picture of the A form? What critical information could not be gleaned from the
A form?

RQ33: What was the difference between the A and B forms of DNA? What information was
derived from the B form (Fig. 1-13b)? Who took the picture of the B form?

RQ34: What was the significance of the fact that the diameter of a DNA molecule is 20 Angstroms?

RQ35: How did Watson and Crick determine how the two chains that make up the helix were held
together?

Reading Assignments and Questions: 2011-2012


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Unit 2: Manipulating and Mapping DNA
(Survey of Biotechnology I)

Text: Ch. 4 (Basic Tools of Recombinant DNA), pp. 75-87

RA1: pp. 75-79 (Beginning of 75…DNA Ligase Joins the Ends)

RQ1: Why is the term “restriction” used in the phrase “restriction nuclease”? What does a
restriction nuclease do?

RQ2: What is a palindromic site? In terms of the structure of most restriction enzymes, why does it
make sense that restriction sites are palindromic?

RQ3: Calculate on average how often a given 5-bp site will occur in a DNA sequence.

RQ4: During electrophoresis, bands containing as little as 0.005 μg of DNA can be visualized.
Express this mass in grams.

RQ5: Why is it significant the DNA molecules can be eluted (recovered) from gels as biologically
intact double helices?

RQ6: What is a restriction map?

RA2: pp. 79-81 (DNA Ligase Joins the Ends…Plasmids and Viruses Are Used as Vectors)

RQ7 (Figure 4-2): Why is it possible to ligate one set of DNA cut with MfeI and another set cut
with EcoRI, even though the restriction sites they recognize are not identical?

RQ8: What is the function of DNA ligase?

RQ9: What were the two basic Asilomar recommendations that were eventually codified into U.S.
law in 1976? How were these laws modified in 1979?

RA3: pp. 81-84 (Plasmids and Viruses Are Used as Vectors...Choosing the Right Starting Material)

RQ10: What is a plasmid?

RQ11: What is a vector?

RQ12: Why does it make sense that antibiotic resistance genes are located on bacterial plasmids
rather than bacterial genomic DNA?

RQ13: What are some of the advantages of pUC18 and pUC19 over some of the earlier plasmids
used in the original cloning experiments?

RQ14 (Figure 4-4): Describe generally two methods for purifying plasmid DNA from bacteria.
Referring to this figure, what does it mean to lyse a cell?
Reading Assignments and Questions: 2011-2012


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RQ15: What is a major advantage of using a bacteriophage vector instead of a plasmid vector?

RQ16: What is cDNA?

RQ17 (Figure 4-5): Describe the five basic steps of cloning DNA in a plasmid.

RA4: pp. 84-87 (Choosing the Right Starting Material…Nucleic Acids Probes Are Used to Locate)

RQ18: Give one example of when it would be appropriate to use cDNA as the starting material for
cloning and another example of when it would be appropriate to use chromosomal (genomic) DNA.

RQ19: What difference is there between mRNA, on the one hand, and ribosomal RNA (rRNA) and
transfer RNA (tRNA), on the other hand, that is exploited to isolate mRNA from total cellular RNA?
How is this done? (See Figure 4-6)

RQ20: How are the poly (A) tails of mRNA used to make a DNA strand from the RNA? (See
Figure 4-7)

RQ21: How is double-stranded DNA (ds-DNA) generated from the RNA-DNA hybrid? (See Figure
4-7)

RQ22: How is the blue/white screening approach used to identify bacteria containing cloned DNA?
Is the evidence of a white or blue colony on an X-gal medium evidence of successful cloning?

RQ23: Why is it necessary to use vectors such as bacterial artificial chromosomes (BACs) and yeast
artificial chromosomes (YACs) to make a genomic library as distinguished from a cDNA library?
(See Figure 4-9)

Reading Assignments and Questions: 2011-2012


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Unit 3: Making DNA
(Survey of Biotechnology II)

Text: Ch. 4 (Basic Tools of Recombinant DNA), pp. 87-92, 94-98

RA1: pp. 87-92 (Nucleic Acid Probes Are Used to Locate…Powerful Methods Are Used to
Sequence

RQ1: Describe generally how a nucleic acid probe can be used to locate a clone carrying a desired
DNA sequence? (See Figure 4-10)

RQ2: Describe the Southern blotting procedure.

RQ3: How does Southern blotting differ from Northern blotting?


RA2: 94-98 (The Polymerase Chain Reaction…PCR Will Amplify Sequences)

RQ4: Approximately how many genes are in the human genome?

RQ5: Until the advent of PCR, techniques designed to isolate and detect rare targets in the genome,
such as genes, were generally complex and time-consuming. The authors write: “PCR changed this
by enabling us to produce enormous numbers of a specified DNA sequence without resorting to
cloning.” Refer back to previous reading questions to describe how you might have gone about
isolating a gene using a cDNA or genomic library prior to the advent of PCR. Then describe how
you would go about the same task using PCR.

RQ6: In PCR, where must the primers be located in relationship to the target sequence?

RQ7: What is the optimal length of a PCR primer?

RQ8 (Figures 4-14 and 15): What are the three fundamental steps of the polymerase chain reaction?

RQ9: What was the single most significant advance in the basic PCR technique?

RQ10: Why is contamination such a problem with PCR?

RQ11: In view of the very real danger of contamination when using PCR, what are two simple,
practical ways to reduce the likelihood of contamination?

RQ12: What is the rate of incorporation of an incorrect nucleotide to a growing DNA chain in PCR
compared to the rate in nature? What accounts for the difference?

RQ13: What is an advantage in using Pfu DNA polymerase over different polymerases in a PCR
reaction?


Reading Assignments and Questions: 2011-2012


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Unit 4: Learning to Read the Book of Life
(Sequencing, Bioinformatics and Gene Discovery)

Text: Ch. 4 (Basic Tools of Recombinant DNA), pp. 92-93; Ch. 5 (Fundamental Features of
Eukaryotic Genes), pp. 107-109, 124; and Ch. 10 (Fundamentals of Whole-Genome
Sequencing), 249-256; 259-263.

RA1: pp. 92-93 (Powerful Methods Are Used to Sequence…End of 93)

RQ1 (Figure 4-12): Describe the Sanger dideoxy DNA sequencing procedure.

RQ2: As of the print date of the text (early 2007), approximately how many non-viral genomes had
been sequenced?

RA2: pp. 259-263 (Fluorescent Labels…Computer Algorithms)

RQ3: Describe the process of automated sequencing using fluorescent labels. Does the underlying
method utilize the dideoxy DNA sequencing concept originally discovered by Fred Sanger? Why is
this method a major advance over using gel slabs?

RA3: pp. 249-252 (Beginning of chapter…The Genomes of Bacteriophages)

RQ4: Why are the amino acid sequences of proteins typically derived from DNA sequencing rather
than from chemical sequencing of purified proteins?

RQ5: What activities constitute the field of genomics?

RQ6: What is the most significant difficulty in sequencing a long, contiguous stretch of DNA vs. a
singe piece of DNA a few hundred base pairs long?

RQ7: What is the basic strategy employed for sequencing long, contiguous stretches of DNA? (See
Figure 10-2)

RA4: pp. 107-109 (Beginning of Chapter…End of Page)

RQ8: Describe the model of the gene proposed by Francois Jacob and Jacque Monod.

RQ9 (Figure 5-1): Study this figure and explain why the orange DNA loops in 5-1(b) do not bind to
the purple strand of RNA.

RQ10: Distinguish between introns and exons. What is the source of the name “exon”?

RQ11: (Figure 5-2): Study the diagram and describe the differences between chromosomal DNA,
the primary RNA transcript (pre-mRNA) and the mature mRNA transcript.

RQ12: Consider the following experiment: Two forms of a Drosophila gene are inserted into the
nuclei of two sets of Xenopus (frog) eggs—one form is unspliced, pre-mRNA (i.e., the pre-mRNA
Reading Assignments and Questions: 2011-2012


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contains introns) and the other form is spliced, mature mRNA (i.e., the introns have been deleted
from the pre-mRNA). The spliced, mature mRNA is much more efficiently transported to the
cytoplasm than the unspliced, pre-mRNA. What does this suggest?

RA5: pp. 124 (What is a gene?)

RQ13: Consider the following facts—many DNA genomic coding sequences are edited and re-
arranged to produce multiple proteins; and some DNA genomic coding sequences code for
functional RNA (such as ribosomal RNA, rRNA, or transfer RNA, tRNA, or small-interfering RNA,
siRNA) but not proteins. Define a gene, taking these facts into account.




Reading Assignments and Questions: 2011-2012


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Unit 5: How Genes Do What They Do
(Gene Regulation and Protein Production)

Text: Ch. 2 (Information Flow from DNA to Protein), pp. 29-44.

RA1: 29-33 (Beginning of Chapter…Mutant Proteins Have Amino Acid Sequences)

RQ1: Give one specific example of each of the following three classes of proteins: structural,
hormonal and enzymatic.

RQ2: Draw a diagram of a dipeptide, using the letter R as a substitute for the specific amino acid
side group, and circling and labeling the peptide bond, the amino end of the peptide and the carboxyl
end of the peptide.

RQ3: How large is the set of amino acids that makes up proteins?

RQ4: How many chains of polypeptides make up most proteins? How many polypeptide chains
make up the hemoglobin molecule?

RQ5: What determines the exact shape of a polypeptide chain? What is the significance of the
shape of the polypeptide chain?

RQ6: Describe the Beadle-Tatum experiment. (See Figure 2-2)

RA2: 33-34 (Mutant Proteins Have Amino Acid Sequences…Genetic Mapping Shows)

RQ7: Describe Vernon Ingram’s 1957 experiment studying sickle cell anemia and the results. How
many more years did it take to identify the cause of the amino acid change from glutamic acid to
valine?

RQ8 (Figure 2-3): Analyze the experiment described in this figure and use the results to explain
how the biosynthetic pathway was determined.

RA3: 34-38 (Genetic Mapping Shows…Messenger RNA Is the Information Carrier)

RQ9: What does it mean to say that genes and their polypeptide products are colinear?

RQ10: Describe Charles Yanofsky’s experiments that established the colinearity of the nucleotide
sequence in genes and their polypeptide products? (Figure 2-4)

RQ11: Show the calculation that groups of two nucleotides could specify 16 amino acids and groups
of four nucleotides 64 amino acids.

RQ12: What is meant by reading frame? Assuming that a codon consists of three nucleotides, what
would happen to the reading frame if one or two bases were added to or deleted from a nucleotide
sequence? What would happen if three bases were added or subtracted from a nucleotide?

Reading Assignments and Questions: 2011-2012


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RQ13: By the early 1960’s, what fact made it obvious that a direct template role for DNA in the
ordering of amino acids was impossible?

RQ14: Why was RNA a good candidate to be the intermediary that transferred information from
DNA to proteins?

RQ15: What is the Central Dogma of molecular biology?

RQ16: What does it mean to make DNA or RNA in a cell-free extract?

RQ17: What are the names of the enzymes that, respectively, make the phosphodiester bonds
between DNA and RNA nucleotides? Identify the parts of a nucleotide on the diagram below.


RQ18: How did the model-building that led Crick and Watson to the structure of DNA in 1953,
probably lead Crick in 1955 to the insight that many, if not all, amino acids had to be attached to
some form of adaptor molecule before they could bind to an RNA template?

RQ19: Which group of molecules was eventually identified as Crick’s proposed adaptor molecule?

RQ20: What is the name of the enzyme that catalyzes the attachment of a specific tRNA to a
specific amino acid? How many of these enzymes exist for the 20 common amino acids?

RA4: 38-42 (Messenger RNA is the Information Carrier… The Basic Steps in Protein Synthesis)

RQ21: What facts led scientists to discard the early hypothesis that ribosomal RNA (rRNA) made
up the RNA templates that ordered amino acids into polypeptide chains?

RQ22: Describe the experiment by Ben Hall and Sol Spiegelman that established that mRNA was
complementary to their associated DNA templates. (Figure 2-8)

RQ23: The end of a gene where transcription begins is conventionally referred to as the 5’ end of
the gene. In fact, is chemical synthesis of a gene initiated at the 5’ or 3’ end of the DNA template?
Why is the transcription start end of a gene referred to as the 5’ end of the gene?

RQ24: What experiment permitted Nirenberg and Matthaei to conclude that the genetic code for
phenylalanine was UUU?

RQ25: What is wobble?
Reading Assignments and Questions: 2011-2012


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RQ26: Distinguish between a sense mutation, non-sense mutation and a missense mutation.

RA5: 42-44 (The Basic Steps in Protein Synthesis…Making Proteins Is a Highly Accurate Process)

RQ27 (Figure 2-11): Describe the process by which proteins are synthesis from the mRNA codon.

RQ27: How do bacterial and yeast RNA polymerases function? What was the experimental basis
for determining how these polymerases function?

RQ28: Compare the speed and accuracy of RNA synthesis with that of DNA synthesis.
Reading Assignments and Questions: 2011-2012


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Unit 6: Finding Genes that Cause Disease
(Linkage and Association Analysis)

Text: Ch. 4 (Basic Tools of Recombinant DNA); pp. 98-100; Ch. 11 (How the Human Genome
Was Sequenced), pp. 280-285; Ch. 14 (Finding Human Disease Genes), pp. 365-381.

RA1: 365-367 (Beginning of Chapter…Recombinant DNA Techniques)

RQ1: Why would it make sense that mutagens might be good candidates for staining various
regions of DNA?

RQ2: What was the key breakthrough in the 1970’s that revolutionized human genetics by
providing a means to identify and clone human genes so that they could be studied in detail?


RA2: 280-285 (Restriction Fragment Length Polymorphisms…End of Page)

RQ3: What are polymorphic DNA markers?

RQ4: What are restriction fragment length polymorphisms?

RQ5: In view of the fact that RFLP’s very rarely are the cause of a genetic disorder, how are they
useful in locating the disorder?

RQ6: What is linkage analysis?

RQ7: Once a marker has been shown to have linkage to a genetic disorder, what needs to be done to
identify the location of the underlying gene on a given chromosome? Describe the coordinated
effort that was undertaken to expedite this step.

RQ8: What are microsatellite markers? Why do they make better DNA markers than restriction
length polymorphisms?

RA3: 98-100 (PCR Will Amplify….Real-Time PCR)


RQ9: Linkage analysis generally requires large pedigrees to demonstrate linkage between two
alleles. Why does linkage analysis of sperm permit linkage analysis based upon the sperm of a
single donor?

RA4: 371-374 (The HGP Revolutionizes…Association Studies)

RQ10: How did the Human Genome Project simplify the process of doing linkage analysis?
Describe the process used to discover the gene responsible for one form of hereditary spastic
paraplegia.

RQ11: What is the basis of the statement that sickle cell disease, although a complex disease with
multiple players, is not a truly complex disorder?
Reading Assignments and Questions: 2011-2012


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RQ12: Why is it not completely accurate to consider susceptibility alleles underlying complex
disorders as “disease genes”?

RA5: 374-376 (Association Studies…Genetic Basis of Alzheimer)

RQ13: Although linkage analysis works well for single-disease disorders, it does not work as well
with complex disorders because the connection between a given disorder and one of the underlying
alleles may not be particularly strong and the relatively small number of individuals involved in a
pedigree (or even the number of variants in a sperm study) is often too small to generate sufficient
statistical evidence of linkage. Describe how a genetic association study differs from linkage
analysis and explain why this is a better technique than linkage analysis for the study of complex
disorders.

RQ14: In an association study, when is a gene suspected to be associated with a disease?

RQ15: In an association study, why is important that members of the case and control groups must
be drawn from the same population? Does this suggest why a population of an isolated group, such
as the population of Iceland, might be particularly useful for association studies?

RQ16: Two different association studies (Short QT Interval and Alzheimer’s Disease) are described
in the text. Use one of these studies as the basis of a paragraph describing how candidate genes were
identified.


RA6: 378-380 (The HapMap Project…Macular Degeneration)

RQ17: What is a haplotype? What is a haplotype block? Why do haploytpes simplify the process
of using SNPs?

RQ18: What is linkage disequilibrium? (See Figure 14-9)

RQ19: What is a “tag” SNP? Approximately how many “tag SNPs” are in the human genome and
how does this number compare with the total number of SNPs?

RQ20: If a positive association were detected between a tag SNP and a given disorder, what would
the next step be in attempting to locate a gene underlying the disorder under study?