Chapter 20, Problem 32ESP

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.

1. (a) How would such a high %T in a primer influence annealing temperature?
2. (b) Compared with oligo(dT) primers, a pool of random sequence primers requires a trickier assessment of annealing temperature. Why?
3. (c) 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?