Review Question - QID 107076

The scientist must convert the mRNA to DNA before any amplification can occur. Only reverse transcriptase can convert mRNA into the complementary DNA (cDNA).

Reverse transcription polymerase chain reaction (RT-PCR) is used to convert RNA into DNA so that it can be amplified. The RT-PCR process begins by adding reverse transcriptase to the solution containing RNA (typically mRNA), dNTPs, primers specific for the sequence of interest, and heat stable DNA polymerase. The RNA is converted into cDNA by the reverse transcriptase protein. Once the cDNA has been formed, it can be used as a template for amplification by PCR. In the clinic, it is used to measure the amount of RNA present in a sample, such as measuring the HIV viral load.

McKay et al. discuss the use of RT-PCR in viral quantification. The amount of RNA can be quantified as relative or absolute. Relative quantification examines the change in the amount of RNA, while absolute quantification provides the exact number of RNA in the sample. While relative quantification is simpler to determine, it requires sequential samples. If no sequential samples are present, absolute quantification is necessary.

Illustration A is a diagram demonstrating the steps in RT-PCR.

Incorrect Answers:
Answer 1: Taq polymerase is used after the cDNA has been made to amplify the sequence.
Answer 2: Ligase is used in molecular cloning to fuse (ligate) two pieces of DNA.
Answer 4: Restriction digestion enzymes are used in molecular cloning to create complementary sticky ends used to bring two different DNA pieces together to be ligated by ligase.
Answer 5: RNA polymerase is only used in the cell to go from DNA to mRNA.