Molecular Biology Techniques Questions and Answers

Q: During in vitro DNA synthesis which of the following radionucleotide is most preferred ?

The preferred label among these is \(^{3}H\) labelled dTTP.When DNA polymerase adds a nucleotide, it attaches the innermost (alpha) phosphate to the growing chain and releases the beta and gamma phosphates as pyrophosphate. A tritium label on the base of dTTP is carried into the new DNA, so the amount of radioactivity in acid-insoluble material measures DNA synthesis.Gamma- and beta-labelled dTTP fail for the reason above: their label leaves in the pyrophosphate and never enters the DNA. (The alpha-labelled nucleotide would work, but it is not offered.) dUTP is not a normal substrate for DNA synthesis, since uracil is excluded from DNA, so carbon-14 labelled dUTP is not the reagent of choice either.

Q: A protein mixture contains three polypeptides A, B and C, whose masses are 63, 28 and 79 kDa with pI values of 6.5, 7.0 and 8.0, respectively, were subjected to standard reducing SDS-PAGE. The order of their separation from top to bottom would be :

From top to bottom the order is C, A and B.SDS coats denatured polypeptides with a uniform negative charge in proportion to their length, and the reducing agent breaks disulfide bonds. Charge differences, and so pI, therefore stop mattering: the gel separates purely by mass, with the smallest polypeptides moving fastest and furthest.So the largest, C (79 kDa), stays nearest the top, then A (63 kDa), and the smallest, B (28 kDa), runs lowest. Orders based on the A, B, C labels or on the pI values (6.5, 7.0, 8.0) apply a criterion that SDS-PAGE ignores.

Q: In a classical experiment, a standard is usesd to :

A standard is used to compare and eliminate nonspecific background noise.In an experiment, a standard or control gives a known reference against which the measured signal is compared. Readings from samples are corrected against it, so that signal arising from the reagents, the instrument or nonspecific binding is removed and only the specific effect remains, as with a blank in spectrophotometry.A standard is never used to add noise. It gives a relative reference rather than recording an absolute signal, and its purpose is to remove nonspecific error, not merely to record it.

Q: What will be the radioactivity of 125I labelled 2 mCi thyroxine after 4 generations (t½ = 60 days) :

The remaining activity is 0.125 mCi.'4 generations' here means four half-lives (4 × 60 = 240 days). Each half-life halves the activity:\[ \begin{aligned} A &= A_0 \left(\tfrac{1}{2}\right)^{4} \\ &= 2 \times \tfrac{1}{16} = 0.125\ \text{mCi} \end{aligned} \]0.25 mCi is the value after three half-lives and 1 mCi after only one. 0.125 µCi has the right number but the wrong unit, a thousand times too small.

Q: Which one of the following is not a fluorophore ?

Acrylamide is not a fluorophore.Acrylamide is the monomer polymerised into polyacrylamide gels. In fluorescence work it is used as a quencher, reducing tryptophan fluorescence to probe how exposed residues are, which is the opposite of emitting light.Acridine orange, quinacrine and ethidium bromide are all fluorescent dyes that bind nucleic acids or chromosomes: ethidium bromide is used to stain DNA in gels, quinacrine to produce Q-bands on chromosomes, and acridine orange to distinguish DNA and RNA.

Q: Which of the following best describes principle of western blotting technique ?

Western blotting is an immunoassay technique preceded by electrophoresis.Proteins are first separated by size on an SDS-polyacrylamide gel, then transferred (blotted) onto a nitrocellulose or PVDF membrane. A specific primary antibody detects the target protein, and a labelled secondary antibody reveals it.Separating restriction fragments by size describes Southern blotting of DNA. Attraction to a specific chemical group describes affinity chromatography. Zero net charge at a particular pH is the isoelectric point, the principle of isoelectric focusing.

Q: Which one produces highest isotope effect ?

The largest isotope effect is produced by tritium, \(^{3}\)H.An isotope effect arises from the difference in mass between isotopes of an element, which changes bond vibration and reaction rates. The effect grows with the relative mass difference:\[ \begin{aligned} ^{3}\text{H} : {}^{1}\text{H} &= 3 \\ ^{14}\text{C} : {}^{12}\text{C} &\approx 1.17 \\ ^{35}\text{S} : {}^{32}\text{S} &\approx 1.09 \\ ^{32}\text{P} : {}^{31}\text{P} &\approx 1.03 \end{aligned} \]Tritium is three times as heavy as ordinary hydrogen, so bonds to it break markedly more slowly. For carbon, sulphur and phosphorus the extra neutrons change the mass by only a few per cent, so their isotope effects are small.

Q: What is the role of β-mercaptoethanol and bromophenol blue used in separation of proteins by SDS-PAGE ?

β-mercaptoethanol is a reducing agent and bromophenol blue a tracking dye.In SDS-PAGE, β-mercaptoethanol reduces disulphide bonds so that proteins unfold fully into separate polypeptide chains, which SDS coats with negative charge. Bromophenol blue is a small negatively charged dye that runs ahead of the proteins and shows the progress of the run, so the gel can be stopped before samples run off.SDS, not mercaptoethanol, is the anionic detergent, and proteins are stained afterwards with Coomassie blue or silver. Mercaptoethanol reduces rather than oxidises. Marker proteins are separate standards of known size, not bromophenol blue.

31.

During in vitro DNA synthesis which of the following radionucleotide is most preferred ?

A.\(^{3}H\) labelled dTTP
B.\(^{14}C\) labelled dUTP
C.\(\gamma\)-\(^{32}P\) labelled dTTP
D.\(\beta\)-\(^{32}P\) labelled dTTP

Exam Year(s)

MH SET - 2023, SET Exam - 2023,

Answer: A

The preferred label among these is \(^{3}H\) labelled dTTP.

When DNA polymerase adds a nucleotide, it attaches the innermost (alpha) phosphate to the growing chain and releases the beta and gamma phosphates as pyrophosphate. A tritium label on the base of dTTP is carried into the new DNA, so the amount of radioactivity in acid-insoluble material measures DNA synthesis.

Gamma- and beta-labelled dTTP fail for the reason above: their label leaves in the pyrophosphate and never enters the DNA. (The alpha-labelled nucleotide would work, but it is not offered.) dUTP is not a normal substrate for DNA synthesis, since uracil is excluded from DNA, so carbon-14 labelled dUTP is not the reagent of choice either.

Discuss in Forum

32.

A protein mixture contains three polypeptides A, B and C, whose masses are 63, 28 and 79 kDa with pI values of 6.5, 7.0 and 8.0, respectively, were subjected to standard reducing SDS-PAGE. The order of their separation from top to bottom would be :

A.A, B and C
B.C, B and A
C.A, C and B
D.C, A and B

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: D

From top to bottom the order is C, A and B.

SDS coats denatured polypeptides with a uniform negative charge in proportion to their length, and the reducing agent breaks disulfide bonds. Charge differences, and so pI, therefore stop mattering: the gel separates purely by mass, with the smallest polypeptides moving fastest and furthest.

So the largest, C (79 kDa), stays nearest the top, then A (63 kDa), and the smallest, B (28 kDa), runs lowest. Orders based on the A, B, C labels or on the pI values (6.5, 7.0, 8.0) apply a criterion that SDS-PAGE ignores.

Discuss in Forum

33.

In a classical experiment, a standard is usesd to :

A.Add background noise
B.Record the absolute value of a signal
C.Compare and eliminate nonspecific background noise
D.Record the non-specific error in the experiment

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: C

A standard is used to compare and eliminate nonspecific background noise.

In an experiment, a standard or control gives a known reference against which the measured signal is compared. Readings from samples are corrected against it, so that signal arising from the reagents, the instrument or nonspecific binding is removed and only the specific effect remains, as with a blank in spectrophotometry.

A standard is never used to add noise. It gives a relative reference rather than recording an absolute signal, and its purpose is to remove nonspecific error, not merely to record it.

Discuss in Forum

34.

What will be the radioactivity of ¹²⁵I labelled 2 mCi thyroxine after 4 generations (t½ = 60 days) :

A.0.25 mCi
B.0.125 mCi
C.0.125 µCi
D.1 mCi

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: B

The remaining activity is 0.125 mCi.

'4 generations' here means four half-lives (4 × 60 = 240 days). Each half-life halves the activity:

\[ \begin{aligned} A &= A_0 \left(\tfrac{1}{2}\right)^{4} \\ &= 2 \times \tfrac{1}{16} = 0.125\ \text{mCi} \end{aligned} \]

0.25 mCi is the value after three half-lives and 1 mCi after only one. 0.125 µCi has the right number but the wrong unit, a thousand times too small.

Discuss in Forum

35.

Which one of the following is not a fluorophore ?

A.Acridine orange
B.Acrylamide
C.Quinacrine
D.Ethidium bromide

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: B

Acrylamide is not a fluorophore.

Acrylamide is the monomer polymerised into polyacrylamide gels. In fluorescence work it is used as a quencher, reducing tryptophan fluorescence to probe how exposed residues are, which is the opposite of emitting light.

Acridine orange, quinacrine and ethidium bromide are all fluorescent dyes that bind nucleic acids or chromosomes: ethidium bromide is used to stain DNA in gels, quinacrine to produce Q-bands on chromosomes, and acridine orange to distinguish DNA and RNA.

Discuss in Forum

36.

Which of the following best describes principle of western blotting technique ?

A.A means of separation of restriction fragments on the basis of size
B.It is based on attraction to a specific chemical group
C.The proteins have a net charge zero at a particular pH
D.It is an immunoassay technique preceded by electrophoresis

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: D

Western blotting is an immunoassay technique preceded by electrophoresis.

Proteins are first separated by size on an SDS-polyacrylamide gel, then transferred (blotted) onto a nitrocellulose or PVDF membrane. A specific primary antibody detects the target protein, and a labelled secondary antibody reveals it.

Separating restriction fragments by size describes Southern blotting of DNA. Attraction to a specific chemical group describes affinity chromatography. Zero net charge at a particular pH is the isoelectric point, the principle of isoelectric focusing.

Discuss in Forum

37.

Which one produces highest isotope effect ?

A.\(^{3}\)H
B.\(^{14}\)C
C.\(^{32}\)P
D.\(^{35}\)S

Exam Year(s)

MH SET - 2025, SET Exam - 2025,

Answer: A

The largest isotope effect is produced by tritium, \(^{3}\)H.

An isotope effect arises from the difference in mass between isotopes of an element, which changes bond vibration and reaction rates. The effect grows with the relative mass difference:

\[ \begin{aligned} ^{3}\text{H} : {}^{1}\text{H} &= 3 \\ ^{14}\text{C} : {}^{12}\text{C} &\approx 1.17 \\ ^{35}\text{S} : {}^{32}\text{S} &\approx 1.09 \\ ^{32}\text{P} : {}^{31}\text{P} &\approx 1.03 \end{aligned} \]

Tritium is three times as heavy as ordinary hydrogen, so bonds to it break markedly more slowly. For carbon, sulphur and phosphorus the extra neutrons change the mass by only a few per cent, so their isotope effects are small.

Discuss in Forum

38.

What is the role of β-mercaptoethanol and bromophenol blue used in separation of proteins by SDS-PAGE ?

A.Anionic detergent and protein staining
B.Reducing agent and tracking dye
C.Oxidising agent and protein staining
D.Reducing agent and marker protein

Exam Year(s)

MH SET - 2025, SET Exam - 2025,

Answer: B

β-mercaptoethanol is a reducing agent and bromophenol blue a tracking dye.

In SDS-PAGE, β-mercaptoethanol reduces disulphide bonds so that proteins unfold fully into separate polypeptide chains, which SDS coats with negative charge. Bromophenol blue is a small negatively charged dye that runs ahead of the proteins and shows the progress of the run, so the gel can be stopped before samples run off.

SDS, not mercaptoethanol, is the anionic detergent, and proteins are stained afterwards with Coomassie blue or silver. Mercaptoethanol reduces rather than oxidises. Marker proteins are separate standards of known size, not bromophenol blue.

Discuss in Forum

Loading…

Topics

Other Subjects

Tags: Molecular Biology Techniques Questions and Answers || Molecular Biology Techniques MCQ Questions and Answers || Molecular Biology Techniques GK Questions and Answers || Molecular Biology Techniques GK MCQ Questions || Molecular Biology Techniques Multiple Choice Questions and Answers || Molecular Biology Techniques GK || GK on Molecular Biology Techniques || Molecular Biology Questions and Answers || Molecular Biology MCQ Questions and Answers || Molecular Biology GK Questions and Answers || GK on Molecular Biology

Molecular Biology Techniques Questions and Answers

Topics

Other Subjects

About Us

Stay informed about all important Dates & Notifications