Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. 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

A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the melting temperature, Tₘ(°C), which is computed by the equation 81.5+0.41×(%GC)−(675/N), where %GC is the percentage of GC nucleotides in the oligonucleotide and N is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer,

5′-TTGAAAATATTTCCCATTGCC-3′

compute the annealing temperature for PCR. What is the relationship between  and %GC? Why? (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.) <>

Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. 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).

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 side 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?