18. Molecular Genetic Tools

Genetic Cloning

Open Question
It 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 Question
A 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 Question
The 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 Question
The 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?
Open Question
To further analyze the CRABS CLAW gene (see Problems 19 and 20), you create a map of the genomic clone. The 11-kb EcoRI fragment is ligated into the EcoRI site of the MCS of the vector shown in Problem 18. You digest the double-stranded form of the genome with several restriction enzymes and obtain the following results. Draw, as far as possible, a map of the genomic clone of CRABS CLAW.

EcoRI 11.0, 3.0   XbaI 4.5, 9.5
EcoRI + XbaI 4.5, 6.5, 3.0   XhoI 13.2, 0.8
EcoRI + XhoI 10.2, 3.0, 0.8   SalI 6.0, 8.0
EcoRI + SalI 6.0, 5.0, 3.0 HindIII 12.0, 1.5, 0.5
EcoRI + HindIII 9.0, 3.0, 1.5, 0.5

What restriction digest would help resolve any ambiguity in the map?
Open Question
To estimate the number of cleavage sites in a particular piece of DNA with a known size, you can apply the formula N/4ⁿ where N is the number of base pairs in the target DNA and n is the number of bases in the recognition sequence of the restriction enzyme. If the recognition sequence for BamHI is GGATCC and the phage DNA contains approximately 48,500 bp, how many cleavage sites would you expect?
Open Question
In Genetic Analysis 14.1 we designed a screen to identify conditional mutants of S. cerevisiae in which the secretory system was defective. Suppose we were successful in identifying 12 mutants.Based on your knowledge of the genetic tools for studying baker's yeast, how would you clone the genes that are mutated in your respective yeast strains? What is an approach to cloning the human orthologs (see Genetic Analysis 14.2 for definition) of the yeast genes?
Open Question
In Genetic Analysis 14.1 we designed a screen to identify conditional mutants of S. cerevisiae in which the secretory system was defective. Suppose we were successful in identifying 12 mutants.Describe the crosses you would perform to determine the number of different genes represented by the 12 mutations.
Open Question
You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene (see Problem 18). You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.

Restriction enzyme sites within a cDNA clone are often also found in the genomic sequence. Can you think of a reason why occasionally this is not the case? What about the converse: Are restriction enzyme sites in a genomic clone always in a cDNA clone of the same gene?
Open Question
You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene (see Problem 18). You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.

Does this tell you anything about where the CRABS CLAW gene is located within the 11-kb genomic clone?
Open Question
In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges (a) 92-26 °C, (b) 45-65 °C, and (c) 65-75 °C.
Open Question
You have identified a 0.80-kb cDNA clone that contains the entire coding sequence of the Arabidopsis gene CRABS CLAW. In the construction of the cDNA library, linkers with EcoRI sites were added to each end of the cDNA, and the cDNA was inserted into the EcoRI site of the MCS of the vector shown in the accompanying figure. You perform digests on the CRABS CLAW cDNA clone with restriction enzymes and obtain the following results. Can you determine the orientation of the cDNA clone with respect to the restriction enzyme sites in the vector? The restriction enzyme sites listed in the dark blue region are found only in the MCS of the vector.