Question

Arrange the steps of diagnosing AIDS using the PCR method in the right order:
I. cDNA, synthesised primers, and deoxyribonucleotides along with buffers are incubated in a thermal cycler.
II. Primers specific to HIV genes are synthesised.
III. Viral RNA is extracted from the patient’s blood cells.
IV. The PCR product is run on a gel to separate and identify the PCR amplified viral genes.
V. Reverse transcriptase is used to convert the RNA to cDNA.

• A. I, III, IV, V, II
• B. IV, I, V, III, II
• C. III, V, II, I, IV
• D. V, II, III, IV, I

Answer 1

The correct option is C III, V, II, I, IV

PCR or polymerase chain reaction is a technique used in molecular biology to amplify a specific DNA segment.

The essential applications include DNA fingerprinting, diagnosing various diseases, detection of pathogens, prenatal diagnosis of diseases, detection of plant pathogens, gene therapy etc.

HIV is a retrovirus (virus with RNA as genetic material) that causes AIDS.

In the PCR method for detecting HIV, a patient's blood cells are isolated and the viral RNA is extracted from it. (III)

Using the RNA as a template, the enzyme reverse transcriptase makes complementary DNA (cDNA). (V)

Thereafter, known genomic sequences of HIV genes are used to synthesise gene-specific primers.(II)

The cDNA, synthesised primers, free deoxyribonucleotides, DNA polymerase and the necessary buffers are mixed in microcentrifuge tubes and incubated in a thermal cycler. (I)

Programmes are set in the cycler in order to adjust the temperature and timings of each of the steps. After the multiple cycles of PCR, the PCR products are run on agarose gel. This separates the amplified DNA (in case the patient is suffering). PCR amplified viral genes are later identified. (IV)

Hence, the correct sequence of steps is III, V, II, I, IV.