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?

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

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?

The U.S. Department of Justice has established a database that catalogs PCR amplification products from short tandem repeats of the Y chromosome (Y-STRs) in humans. The database contains polymorphisms of five U.S. ethnic groups (African-Americans, European Americans, Hispanics, Native Americans, and Asian-Americans) as well as the worldwide population.

For forensic applications, the probability of a 'match' for a crime scene DNA sample and a suspect's DNA often culminates in a guilty or innocent verdict. How is a 'match' determined, and what are the uses and limitations of such probabilities?

The U.S. Department of Justice has established a database that catalogs PCR amplification products from short tandem repeats of the Y chromosome (Y-STRs) in humans. The database contains polymorphisms of five U.S. ethnic groups (African-Americans, European Americans, Hispanics, Native Americans, and Asian-Americans) as well as the worldwide population.

What would be the value of knowing the ethnic population differences for Y-STR polymorphisms?

The U.S. Department of Justice has established a database that catalogs PCR amplification products from short tandem repeats of the Y chromosome (Y-STRs) in humans. The database contains polymorphisms of five U.S. ethnic groups (African-Americans, European Americans, Hispanics, Native Americans, and Asian-Americans) as well as the worldwide population.

Y-STRs from the nonrecombining region of the Y chromosome (NRY) have special relevance for forensic purposes. Why?