Open Question
Outline the roles played by restriction enzymes and vectors in cloning DNA.
18. Molecular Genetic Tools
Genetic Cloning
Back18. Molecular Genetic Tools
Genetic Cloning
- Open Question
In 1975, the Asilomar Conference on Recombinant DNA was organized by Paul Berg, a pioneer of recombinant DNA technology, at a conference center at Asilomar State Beach in California. Physicians, scientists, lawyers, ethicists, and others gathered to draft guidelines for safe applications of recombinant DNA technology. These general guidelines were adopted by the federal government and are still in practice today. Consider the implications of recombinant DNA as a new technology. What concerns might the scientific community have had then about recombinant DNA technology? Might those same concerns exist today?
- Open QuestionWhy are diseases of the blood simpler targets for treatment by gene therapy than are many other genetic diseases?
- Open QuestionInjection of double-stranded RNA can lead to gene silencing by degradation of RNA molecules complementary to either strand of the dsRNA. Could RNAi (see Sections 13.3 and 14.3) be used in gene therapy for a defect caused by a recessive allele? A dominant allele? If so, what might be the major obstacle to using RNAi as a therapeutic agent?
- Open QuestionWhat are some of the impacts of biotechnology on crop plants in the United States?
- Open Question
In the context of recombinant DNA technology, of what use is a probe?
- Open QuestionSummarize the arguments for and against patenting genetically modified organisms.
- Open Question
If you performed a PCR experiment starting with only one copy of double-stranded DNA, approximately how many DNA molecules would be present in the reaction tube after 15 cycles of amplification?
- Open QuestionIt is often desirable to insert cDNAs into a cloning vector in such a way that all the cDNA clones will have the same orientation with respect to the sequences of the plasmid. This is referred to as directional cloning. Outline how you would directionally clone a cDNA library in the plasmid vector pUC18.
- Open QuestionA major advance in the 1980s was the development of technology to synthesize short oligonucleotides. This work both facilitated DNA sequencing and led to the advent of the development of PCR. Recently, rapid advances have occurred in the technology to chemically synthesize DNA, and sequences up to 10 kb are now readily produced. As this process becomes more economical, how will it affect the gene-cloning approaches outlined in this chapter? In other words, what types of techniques does this new technology have potential to supplant, and what techniques will not be affected by it?
- Open QuestionThe bacteriophage lambda genome can exist in either a linear form (see Figures 15.1 and 15.8) or a circular form.How many fragments will be formed by restriction enzyme digestion with XhoI alone, with XbaI alone, and with both XhoI and XbaI in the linear and circular forms of the lambda genome?
- Open Question
You have recovered a cloned DNA segment from a vector and determine that the insert is 1300 bp in length. To characterize this cloned segment, you isolate the insert and decide to construct a restriction map. Using enzyme I and enzyme II, followed by gel electrophoresis, you determine the number and size of the fragments produced by enzymes I and II alone and in combination, as recorded in the following table. Construct a restriction map from these data, showing the positions of the restriction-enzyme cutting sites relative to one another and the distance between them in units of base pairs.
Enzyme Restriction Fragment Sizes (bp)
I 350, 950
II 200, 1100
I and II 150, 200, 950 - Open Question
The restriction enzymes XhoI and SalI cut their specific sequences as shown below:
Xhol 5'-C TCGAG-3'
3'-GAGCT C-5'
Sall 5'-G TCGAC-3'
3'-CAGCT G-5'
Can the sticky ends created by XhoI and SalI sites be ligated? If yes, can the resulting sequences be cleaved by either XhoI or SalI? - Open Question
The bacteriophage ϕX174 has a single-stranded DNA genome of 5386 bases. During DNA replication, double-stranded forms of the genome are generated. In an effort to create a restriction map of ϕX174, you digest the z-stranded form of the genome with several restriction enzymes and obtain the following results. Draw a map of the ϕX174 genome.
Pstl 5386 PstI + PsiI 3078, 2308
Psil 5386 PstI + DraI 331, 1079, 3976
Dral 4307, 1079 PstI + DraI 898, 1079, 3409 - Open QuestionThe Age of Genetics was created by remarkable advances in the use of biotechnology to manipulate plant and animal genomes. Given that the world population reached 7.5 billion people in 2017 and is expected to reach 9.7 billion in 2050, some scientists have proposed that only the worldwide introduction of genetically modified (GM) foods will increase crop yields enough to meet future nutritional demands. Pest resistance, herbicide, cold, drought, and salinity tolerance, along with increased nutrition, are seen as positive attributes of GM foods. However, others caution that unintended harm to other organisms, reduced effectiveness to pesticides, gene transfer to nontarget species, allergenicity, and as yet unknown effects on human health are potential concerns regarding GM foods. If you were in a position to control the introduction of a GM primary food product (rice, for example), what criteria would you establish before allowing such introduction?