Question

Arrange the steps of diagnosing AIDS using the RT-PCR method in the right order.

I. DNA, engineered primers (specific to HIV genes), and nucleotides along with buffers are incubated in a thermal cycler.
II. Viral RNA is extracted from the patient’s plasma or blood sample.
III. The PCR product is run on a gel to separate and identify the PCR amplified viral genes.
IV. Reverse transcriptase is used to convert the RNA to cDNA.

• A. I → II → IV → III
• B. IV → I → III → II
• C. II→ IV → I → III
• D. II → III → IV → I

Answer 1

The correct option is C II→ IV → I → III

RT-PCR is a highly sensitive technique which is used for detection and quantification of RNA. PCR does not work on RNA directly. Hence in RT-PCR, the RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase, and then PCR is used to amplify the cDNA (also known as copy DNA).

AIDS is caused by HIV, which is a retrovirus (viruses containing RNA as genetic material).

RT-PCR is used for its detection as it amplifies even a single copy of RNA to produce millions of copies of cDNA.

For detecting HIV, a presumptive patient’s plasma is isolated and the viral RNA is extracted from it.

Using the RNA as a template and the enzyme reverse transcriptase, RNA is reverse-transcribed to cDNA molecules, which is single stranded.

Thereafter, known RNA sequences of essential HIV genes are used to synthesize gene specific primers.

Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length. They serve as the starting point for the synthesis of DNA.

The reverse transcribed cDNA, the synthesized primers, free nucleotides and the necessary buffers are mixed and incubated in a thermal cycler.

After the multiple cycles of PCR, the PCR products are identified via agarose gel electrophoresis, which separates the bands of amplified cDNA according to their size, under the influence of electric field. The bands of separated DNA are analysed further to detect presence of pathogens in the patient’s plasma.

Hence, steps of diagnosing AIDS using the RT-PCR method can be summarised as follows:

Viral RNA is extracted from the patient’s plasma or blood sample (II).
Reverse transcriptase is used to convert the RNA to cDNA (IV).
DNA, engineered primers, and nucleotides along with buffers are incubated in a thermal cycler (I).
The PCR product is run on a gel to separate and identify the PCR amplified viral genes (III).