18. Molecular Genetic Tools

The restriction enzymes XhoI and SalI cut their specific sequences as shown below:

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?

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.

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?

To further analyze the CRABS CLAW gene, 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.

What restriction digest would help resolve any ambiguity in the map?

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 $\lambda$ phage DNA contains approximately 48,500 bp, how many cleavage sites would you expect?

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 of the yeast genes?

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.

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?

You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene. 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?

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

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.

We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?

Traditional Sanger sequencing has largely been replaced in recent years by next-generation and third-generation sequencing approaches. Describe advantages of these sequencing methods over first-generation Sanger sequencing.

Given your knowledge of the genetic tools for studying Drosophila, outline a method by which you could clone the dunce and rutabaga genes identified by Seymour Benzer's laboratory in the genetic screen.

How is fluorescent in situ hybridization (FISH) used to produce a spectral karyotype?