Question

Q2 A mutant monkey was identified that showed resistance to a viral disease ‘Kansah’. The kan gene, responsible for the disease resistance, was identified and cloned. A molecular technique revealed the presence of kan in human cells. Later these researchers isolated RNA from liver, kidney, gut and skin cells. The researchers prepared cDNA using reverse transcriptase and then performed real-time PCR to measure the expression of the kan gene. The Ct values for samples from gut, kidney, liver and skin cells were 20, 25, 23 and 38, respectively. Answer the following questions

a. What is real-time PCR? How does it differ from the standard PCR?

b. What do you understand by the term Ct value? Explain using a figure.

c. Which cells have the highest expression of kan? Explain your answer.

d. If a liver cell makes 1,000 molecules of Kan protein, how many molecules will you expect from a single kidney cell?

e. If a liver cell makes 1,000 molecules of Kan protein, how many molecules will you expect from a single gut cell?

Answer 1

Ansa) Real time PCR also known as quantitative RT PCR is a method that is based on general principle of PCR, which is used to amplify and simultaneously quantify a target DNA molecule.RT PCR allowed  the scientist or researchers to actually visualize the increase in the amount of DNA as it is amplified that's why it is called as real time PCR.

Real time PCR system based upon the detection and quantification of a fluorescent reporter whose signal increases in direct proportional to the amount of target DNA ie PCR product in a reaction. These fluorescent reporter molecules include dyes that bind to the double stranded DNA or sequence specific probes..

Standard PCR method used to amplify target DNA while in real time PCR the target DNA is amplified as well as amplified DNA quantified as it is amplified. In standard PCR technique amplification is detected at the end of reaction by amplified sample on  gel electrophoresis while in case of real time PCR amplification results are detected at early stage of reaction.

Real time pcr is more sensitive than standard PCR.

Ansb) ct value stands for cycle threshold value and defined as the number of cycles that are required by fluorescent signal for crossing the the threshold value .

Ct value is inversely proportional to the amount of target DNA in PCR sample.

Ans c) gut cell have highest expression of kan protein because it has least ct value ie, 20 as compare to other cells which indicate more amount of target DNA in gut cell which code for kan protein so more kan protein expression.

Ansd) ct value of liver cell x molecules of kan inliver  cell=ct value of kidney cell x molecules of kan in kidney

Molecules of kan in kidney=23x1000/25=920 molecules of kan protein

No of kan protein in kidney cell=920

Anse) No of kan protein molecules in gut cell=ct value of liverx no of kan molecule in liver/ kt value of gut=23x1000/20=1150 molecules

No of kan protein in gut cell =1150