This set of Genetic Engineering Questions and Answers for Experienced people focuses on “Modifications in PCR – 2”.
1. Which one of the following is not done if amplification of the non flanking region is carried out?
a) Firstly, restriction enzyme digestion is done of those sites whose sequence is not known
b) Then the self-ligation of molecules is allowed
c) Now the molecules are cleaved where the known sequence is
d) Again, the molecules are linearized and the known sequence is in the middle
Explanation: If such amplification has to be carried out, firstly the restriction enzyme digestion is done at those sites whose sequence is not known till now. Then self ligation is carried out i.e. conditions for intramolecular ligation is applied. As the molecules circularize now, cleavage is carried out of known sequence. As the known sequence is cleaved, the molecule is now cleaved with a known sequence at the ends. As it known sequence, primers can be constructed for it and the unknown region is amplified.
2. Reverse transciptase PCR is also carried out at times. Which of the statement is true?
a) Amplification of RNA samples is not required for knowing the abundance of mRNA
b) Both the start and the end primers are used
c) Only a single cDNA strand is synthesized before the PCR
d) The primer used is always specific
Explanation: Reverse transciptase PCR is carried out by the use of reverse transciptase enzyme. RNA amplification is necessary at times to know the abundance of mRNA. In this only one primer is used and one cDNA strand is synthesized before the PCR. The primer can be oligo-dT for general cDNA synthesis or a specific primer is used.
3. Which type of PCR allows the use of permeabilized tissue?
a) Inverse PCR
b) In-situ PCR
c) Quantitative PCR
d) Hot-start PCR
Explanation: In-situ PCR allows the use of permeabilized tissue, such as thin sections on a microscopic slide. The PCR product is detected by hybridization and it allows locating the nucleic acid in the target tissue.
4. How many approaches are there for measuring the quantity of PCR products?
Explanation: There are basically two approaches for measuring the amount of PCR products. These are known as end-point approach and real time approach. In end-time approach, the amount is measured once the PCR is done. In real time approach the amount is measured while the PCR reaction is still going on.
5. Which of the statement hold true for Quantitative PCR?
a) A fluorescent dye is used which binds on single stranded DNA molecules
b) SYBR green is an example such type of dye
c) The quantity of DNA is simply measured by measuring the amount of fluorescence
d) This approach is useful if the products are non-specific in nature
Explanation: A fluorescent dye is used which binds on double stranded DNA molecules and SYBR green is an example of such type of dye. The quantity of DNA is measured by simply measuring the amount of fluorescence. It is used only in the case if products are created specifically because it simply relies on binding of the dye to double stranded molecules.
6. In another method of quantitative PCR, reporter-quencher set up is used. Which of the statement holds true for this methodology?
a) It allows detection of all double stranded molecules
b) The reporter and quencher are the molecules present on the same probe
c) The quencher is having a fluorescent group
d) Fluorescence is observed only when both the groups are present in proximity to each other
Explanation: This method allows the detection of only specific molecules. It is so because the probes are specific for areas to be amplified. The probe consists of reporter at one end and quencher at the other. The reporter consists of the fluorescent group which fluoresce only when the quencher is released. Quencher is released as a specific binding of the probe is taking place.
7. By using two oligonucleotides, we can measure the amount of quantity of DNA by measuring the amount of fluorescence. The first probe absorbs light and the second emits it at a particular wavelength.
Explanation: If two oilgonucleotides are used, which bind nearby to the target DNA help in quantifying the DNA. The first probe is meant for absorbing light and the second probe absorbs the light emitted by the first one and then re-emits it. DNA is quantified by measuring the fluorescence from the second probe and it is possible only when both the probes are close to each other i.e. they have bound to the location near the target DNA.
8. Choose the correct statement with regard to quantitative PCR?
a) End-point PCR is favourable over real time PCR
b) In real time PCR, quantification is done as the reaction is going on
c) If the product measurement is done after the completion then the measurement is done by target sequence and no other factor affects it
d) If the primers are available in limited amount, then the product obtained is proportional to the target sequence
Explanation: Real-time PCR is favourable over end-point PCR. It is so because real-time PCR is performed when the PCR is going on and the end-point PCR is performed when the PCR has been completed. In the end-point PCR, it is limited by the amount of reactants such as primers. If fewer amounts of primers are there, the product formed is less and is not proportional to the amount of target sequence only.
9. Mutation is never deliberately induced in PCR products.
Explanation: Mutation is deliberately induced in PCR product at times and the process is termed as mutagenesis. In mutagenesis, primers are constructed in such a way that they correspond to the mutated sequence rather than the original one.
10. If amplification of one of the strand is favoured, the modification of PCR is known as __________
a) single-strand PCR
b) partial PCR
c) asymmetric PCR
d) anchored PCR
Explanation: If the amplification of one of the strand is preferred, it is called asymmetric PCR. This leads to the formation of single stranded products which are having uses such as in sequencing.
11. Which of the statement is incorrect for anchored PCR?
a) Anchored PCR is the modification in which only one piece of the sequence is known and thus one primer
b) The known sequence is attached to the required region of amplification and then further used as a second priming site
c) Fragment the sample DNA and ligate it to known sequence
d) Tails are added enzymatically to the region of known sequence
Explanation: Anchored PCR is based on the fact that only a piece of the sequence is known and thus only one primer. The known sequence is attached to the required region of amplification and is further used as a second priming site. The sample DNA is fragmented and ligated to known sequence. Also, enzymatically tails are added to the sample DNA or to the molecules formed after first round of synthesis. This tail can be further used as a primer.
12. Emulsion or droplet PCR is another modification of PCR. Which of the statement holds true?
a) It is possible to incorporate the reagents in a lipid drop
b) It refers to the PCR carried out at large scale
c) The temperature variation is not possible easily
d) If there is a single template molecule at the start then amplification results in the mixture and it because of the extent of extension carried out
Explanation: It is possible to incorporate the reagents in a lipid drop. In this type, PCR is carried out at a small scale. The temperature of these drops can be varied easily. If there is a single template in the DNA molecule then the amplification results in only one type of molecules.
13. Isothermal amplification is carried out at times. Which of the statement is true?
a) The repeated heating and cooling required by PCR is not the reason for limiting how quickly the reaction is carried out
b) It is carried out typically at 65 degrees
c) Normal Taq polymerase is used
d) It requires expensive PCR machines to be carried out
Explanation: Isothermal PCR is that in which the reaction is carried out at typically 65 degrees. The repeated heating and cooling cycles required by the PCR is the reason for limiting how quickly the reaction is carried out. In this case, DNA polymerase with strand displacing activity is used and it avoids heating at high temperatures. It is a rapid process and expensive PCR machines are not used.
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