About 1% of occurrences of nonautoimmune type 1 diabetes are due to loss-of-function alleles in the insulin gene. Individuals heterozygous for such mutations develop diabetes as infants or in the first few years of their lives. Outline how you might approach gene therapy for such a disease and what difficulties you might encounter.

You have cloned a gene for an enzyme that degrades lipids in a bacterium that normally lives in cold temperatures. You wish to transfer this gene into E. coli to produce industrial amounts of enzyme for use in laundry detergent.

You have managed to produce transgenic E. coli expressing mRNA of your gene, but only a low level of protein is produced. Why might this be so? How could you overcome this problem?

You have cloned a gene for an enzyme that degrades lipids in a bacterium that normally lives in cold temperatures. You wish to transfer this gene into E. coli to produce industrial amounts of enzyme for use in laundry detergent.

How would you accomplish this?

Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

Given that formamide competes for hydrogen bond locations on nucleic acid bases and monovalent cations are attracted to the negative charges on nucleic acids, explain why the Tₘ varies as described in part (a).

Most of the techniques (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

For the following concentrations of Na⁺ and formamide, calculate the Tₘ. Assume 45% GC content.

  [Na⁺]  % Formamide

  0.825      20

  0.825      40

  0.165      20

  0.165      40

In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR), Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.

If one were interested in comparing the quantitative distribution of gene expression in, say, the right and left sides of a developing heart, how might one proceed using RT-PCR?

In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR), Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.

Compared with oligo(dT) primers, a pool of random sequence primers requires a trickier assessment of annealing temperature. Why?

In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR), Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.

How would such a high %T in a primer influence annealing temperature?

Describe how having the Cas9 gene at a genomic locus unlinked to the guide RNA and target site locus in an engineered gene drive system could slow the propagation of the gene drive allele in a population into which a small number of individuals carrying both the gene drive allele and the Cas9 locus are released.