Q&A Hero

Q: Reverse transcriptase is often used as the heat-stable enzyme in PCR.

Q: A restriction map provides the location of sites cleaved by restriction enzymes.

Q: In recombinant DNA technology, YAC, RFLP, and λ have identical uses.

Q: E. coli is a common YAC.

Q: In recombinant DNA technology, a YAC is an enzyme isolated from a large South American, four-legged mammal.

Q: Some restriction endonucleases are capable of producing blunt ends; others can generate "sticky" ends.

Q: To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.

Q: In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.

Q: Restriction endonucleases typically recognize palindromic DNA sequences and often generate "sticky ends" or single-stranded DNA overhangs at cut sites.

Q: A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.

Q: What method might be used to study a knockout mouse that, by virtue of the lost gene or genes, generates a lethal condition?

Q: What is a popular approach that is often used to introduce the targeting vector into cells?

Q: What is the fundamental purpose of creating a knockout organism?

Q: Assume that you have cut λ DNA with the restriction enzyme HindIII. You separate the fragments on an agarose gel and stain the DNA with ethidium bromide. You notice that the intensity of the stain is less in the bands that have migrated closer to the "+" pole. Give an explanation for this finding.

Q: Nucleic acid blotting is commonly used in molecular biology. Two types, Southern blots and northern blots, involve gel electrophoresis and a filter, which holds the nucleic acid. Briefly describe the procedure of "blotting" in this context and differentiate between Southern and northern blots.

Q: In what way are specific DNA sequences of the template amplified in the polymerase chain reaction? In other words, how does one target the target?

Q: In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?

Q: What is the name of the process by which bacterial colonies (cells) are transferred from one agar plate to another, maintaining the same spatial pattern?

Q: What is the specific application of reverse transcriptase in the preparation of cDNA?

Q: What is a cDNA molecule?

Q: Under ideal conditions, how many copies of all the sequences of the host genome should be represented in a genomic library?

Q: Following are four processes common to most cloning experiments: a) transforming bacteria b) plating bacteria on selective medium c) cutting DNA with restriction endonucleases d) ligating DNA fragments Place components of this list in the order in which they would most likely occur during a cloning experiment.

Q: If one wishes to clone a gene using typical restriction endonucleases, how does the restriction endonuclease recognize genes in the genome?

Q: When propagating a clone in the lambda phage, would you have more immediate success if the phage entered the lysogenic or the lytic cycle?

Q: Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?

Q: Of what advantage is it to have a multiple cloning site (multiple unique restriction sites) embedded in the lacZ component of a plasmid?

Q: What term is used to refer to the process in which DNA can be introduced into host bacterial cells?

Q: Name at least two typical characteristics of a DNA cloning plasmid?

Q: What is meant by the designation EcoRI?

Q: What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?

Q: List, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.

Q: Over the years, sophisticated plasmid vectors have been developed for use in recombinant DNA technology. List at least two features that have been introduced in particularly useful vectors.

Q: Some restriction enzymes cleave DNA in such a manner as to produce blunt ends. Most often ligation of blunt end fragments is enhanced by the use of the enzyme terminal deoxynucleotidyl transferase. Speculate on the function of deoxynucleotidyl transferase in terms of using blunt end fragments in cloning.

Q: Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000. Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?

Q: Molecular biologists rely on many, often sophisticated, techniques to pursue their discipline. One may list ultracentrifugation, electron microscopy, X-ray diffraction, electrophoresis, and computer interfacing as fundamental tools. Model organisms provide the raw materials for study. List three "organisms" (or organismic groups) often used by recombinant DNA technologists and describe a major advantage of each group.

Q: In the context of recombinant DNA technology, what is meant by the term vector?

Q: What is recombinant DNA technology? What would you list as safety issues associated with recombinant DNA technology?

Q: A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.A) OH; 2-²; 3-²B) methyl; 2-²; 3-²C) carboxyl; 5-²; 3-²D) OH; 2-²; 5-²E) None of the above is correct.

Q: In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates)? A) citric acid cycle B) DNA sequencing C) restriction digestion D) electron transport E) plasmolysis

Q: Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________. A) hybridizes filter-bound DNA with a DNA probe B) hybridizes filter-bound RNA with a DNA probe C) examines amino acid substitutions with radioactive probes D) cleaves RNA with restriction endonucleases E) ligates DNA with DNA ligase

Q: The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95oC. Why would such a heat-stable polymerase be beneficial in PCR? A) Each cycle includes a "hot" saturation phase (95C), which allows the primers to anneal to the target DNA. B) Each cycle includes a "hot" denaturation phase (95C), which serves to sterilize the culture. C) Each cycle includes a "hot" denaturation phase (95C), which activates the Taq polymerase. D) Each cycle includes a "hot" denaturation phase (95C), which separates the hydrogen bonds that hold the strands of the template DNA together. E) More than one of the above are correct.

Q: In the context of molecular genetics, reverse transcription PCR (RT-PCR) refers to ________.A) assembling a DNA sequence from an mRNAB) assembling an RNA sequence from a DNA sequenceC) translating in the 3-² to 5-² directionD) transcribing first, then translatingE) making an amino acid sequence from a DNA sequence

Q: Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion. A) 400, 800, 1000 (2 of these) B) 400, 1200, 1600 C) 300, 700, 2200 D) 700, 400, 1400, 2600 E) 300, 700, 1000, 1200

Q: Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term is given to this advantageous arrangement of restriction sites? A) palindrome B) consensus sequence C) multiple cloning site D) β-galactosidase E) complementation

Q: List two especially useful characteristics of cloning vectors. A) high copy number and antibiotic resistance gene(s) B) virulence and lysogenicity C) ability to integrate into the host chromosome and then causing a lytic cycle D) nonautonomous replication and transposition E) reverse transcriptase and ligase activities

Q: Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky? A) single-stranded complementary tails B) blunt ends C) poly-A sequences D) 5"² cap E) interference

Q: Words such as level, civic, and kayak have something in common compared to the fundamental tool of recombinant DNA technology. In the context of recombinant DNA technology, which term would be used to describe such words? A) lysogenic B) prototrophic C) palindromic D) conjugation E) insertion

Q: A retrovirus uses reverse transcriptase to make a DNA copy of RNA.

Q: When referring to tumor-suppressor genes and cancer, loss of heterozygosity is likely to suppress cancer formation.

Q: When the normal retinoblastoma protein is dephosphorylated, it acts to suppress cell division by binding to and inactivating the E2F transcription factor.

Q: There are two types of retinoblastoma, familial and sporadic. In the familial form, one generally inherits a defective gene from one parent.

Q: A tumor-suppressor gene normally functions to suppress cell division.

Q: The gene p53 is called the "guardian of the genome" because it corrects mutations in the spindle apparatus before nondisjunction can occur.

Q: There are several checkpoints in the mitotic cell cycle. All occur in the S phase.

Q: Any agent that causes damage to DNA is a potential carcinogen.

Q: The genome of humans is remarkably stable, so much so that there are no cancers known to result from genomic instability.

Q: As more is learned about cancer, it has become clear that cancer, with few exceptions, has no genetic basis.

Q: Provide a simple definition of a carcinogen.

Q: List at least three environmental agents or factors that are known to cause cancer.

Q: In what way might a virus contribute to cancer formation?

Q: The familial form of retinoblastoma is characterized by cancer appearing in both eyes relatively early in life. In contrast, the sporadic form is usually unilateral and appears later. Why the difference?

Q: The genetic difference between familial retinoblastoma and sporadic retinoblastoma appears to be based on those with the familial form starting out being ________, whereas those with the sporadic form start out being ________.

Q: In what way can loss of heterozygosity lead to cancer?

Q: If someone has a predisposition to cancer, what genetic circumstance likely exists?

Q: Name three human cancers with a genetic predisposition. What appears to be the genetic cause of each?

Q: What are two properties that various types of cancer cells share?

Q: Much has been written about p53 in terms of cancer biology. What is p53, and what is its significance?

Q: Differentiate among the following types of genes: tumor-suppressor gene, proto-oncogene, and oncogene.

Q: Describe the molecular nature of mutation, as related to cancer, in a ras gene.

Q: List three general categories of genetic changes that lead to the formation of oncogenes.

Q: What is the name of a normal gene that serves to promote cellular division?

Q: Describe three genetic mechanisms whereby proto-oncogenes can become overexpressed.

Q: Describe the cellular and molecular function of the ras gene family and the consequences of mutations in ras.

Q: What is a tumor-suppressor gene? What are oncogenes? What is the normal (nonmutant) cellular version of an oncogene called?

Q: What is retinoblastoma, and what is its supposed genetic basis?

Q: Describe the general relationship that may exist between mutations and cancer.

Q: Name two of the classes of proteins that combine to directly control progression through the cell cycle.

Q: Describe two classes of proteins known to be involved in the regulation of the cell cycle.

Q: Which three stages or transitions in the cell cycle seem to serve as points of control (checkpoints)?

Q: What functional differences exist between various cyclins?