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?

Key Concepts

Reverse Transcription PCR (RT-PCR)

RT-PCR is a laboratory technique used to convert RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This process allows researchers to amplify specific RNA sequences, making it easier to study gene expression. In the context of congenital heart disease, RT-PCR can help identify mutations affecting gene function by analyzing the expression levels of relevant genes in heart tissue.

Annealing Temperature

The annealing temperature is the temperature at which primers bind to their complementary DNA sequences during PCR. It is crucial for the specificity and efficiency of the amplification process. A higher percentage of thymine (T) in a primer can lower the melting temperature (Tm), which may require adjustments to the annealing temperature to ensure optimal binding and reduce non-specific amplification.

Oligo(dT) Primers

Oligo(dT) primers are short sequences of deoxythymidine that bind to the poly-A tail of mRNA molecules. They are commonly used in RT-PCR to selectively reverse transcribe mRNA into cDNA, allowing for the study of gene expression. The high thymine content in these primers influences their binding properties and the resulting annealing temperature, which is critical for successful amplification of the target sequences.