Question

Arrange the steps of diagnosing AIDS using the PCR method in the right order:
I - cDNA, engineered primers, and nucleotides along with buffer are incubated in a thermal cycler
II - Primers specific to HIV genes are engineered
III - RNA is extracted from the patient’s plasma
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

In the PCR method for detecting HIV, a presumptive patient’s plasma is isolated and the RNA is extracted from it. Using the RNA as a template, the enzyme reverse transcriptase reverse-transcribes the RNA to a cDNA or a complementary DNA molecule. Thereafter, known RNA sequences of essential HIV genes like gag, poly and coat are used to synthesize gene specific primers. The transcribed cDNA, the synthesized primers, free nucleotides and the necessary buffers are mixed in microcentrifuge tubes 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 DNA according to size. The bands of DNA are compared against a known ladder to deduce the molecular weight (MW) of the viral genes, if present in the patient’s plasma.