BIO203 — Methods in Molecular Biology: Midterm & Finalterm Solved MCQs and Subjective

BIO203 Methods in Molecular Biology covers agarose gel electrophoresis, PCR and its types, restriction enzymes and cloning, DNA extraction and quantification, transformation, blotting and SDS-PAGE. This solved post gives you 30 verified items for Virtual University of Pakistan: 10 Midterm MCQs from Lectures 1-22, 10 Finalterm MCQs from Lectures 23-45, and 5 Midterm plus 5 Finalterm subjective questions, each with its correct answer and handout reference. Practice every question instantly in the quiz for this course. Take the quiz: https://virtualustaad.blogspot.com/p/solvedmcqs.html?code=BIO203

1. Midterm Solved MCQs

Q1. Agarose, which is used to make gels for electrophoresis, is extracted from: A) Bacteria B) Seaweeds (red algae) C) Fungi D) Land plants.
Correct answer: B — Seaweeds (red algae).
Reference: VU handout — Agarose gel electrophoresis.

Q2. In PCR, the denaturation step (separation of the DNA double helix) takes place at about: A) 47 °C B) 54 °C C) 60 °C D) 94 °C.
Correct answer: D — 94 °C.
Reference: VU handout — Polymerase chain reaction.

Q3. In agarose gel electrophoresis, DNA fragments migrate toward the: A) Negative electrode (cathode), because DNA is positively charged B) Positive electrode (anode), because DNA is negatively charged C) Negative electrode, because DNA is neutral D) Neither electrode; DNA does not move in an electric field.
Correct answer: B — Positive electrode (anode), because DNA is negatively charged.
Reference: VU handout — Agarose gel electrophoresis.

Q4. DNA stained with ethidium bromide can be seen: A) In visible light without any treatment B) Only after staining with Coomassie blue C) Under exposure to UV light D) Only under an electron microscope.
Correct answer: C — Under exposure to UV light.
Reference: VU handout — Agarose gel electrophoresis; visualisation of DNA.

Q5. Restriction endonucleases cut DNA: A) At random positions B) Only at the ends of the DNA molecule C) At specific recognition sequences (usually palindromic) D) Only in RNA, not DNA.
Correct answer: C — At specific recognition sequences (usually palindromic).
Reference: VU handout — Restriction digestion of DNA.

Q6. T4 DNA ligase uses which molecule as its energy source when it seals nicks in DNA? A) NAD+ B) ATP C) GTP D) Acetyl-CoA.
Correct answer: B — ATP.
Reference: VU handout — Recombinant DNA; cloning.

Q7. In PCR, the annealing temperature is typically in the range of: A) 94-98 °C B) 72 °C C) 50-65 °C D) 37 °C.
Correct answer: C — 50-65 °C.
Reference: VU handout — Polymerase chain reaction.

Q8. Reverse transcription is the process of: A) Synthesising RNA from a DNA template B) Synthesising DNA (cDNA) from an RNA template C) Translating mRNA into protein D) Copying DNA into DNA.
Correct answer: B — Synthesising DNA (cDNA) from an RNA template.
Reference: VU handout — Reverse transcription; cDNA synthesis.

Q9. An antibiotic-resistance gene in a cloning vector is mainly used for the selection of: A) Competent bacterial cells B) Transformed bacterial cells (those that have taken up the plasmid) C) Recombinant cells only, excluding non-recombinant transformants D) Cells that have lost the plasmid.
Correct answer: B — Transformed bacterial cells (those that have taken up the plasmid).
Reference: VU handout — Cloning vectors; selection of transformants.

Q10. In agarose gel electrophoresis, compared with larger DNA fragments, smaller DNA fragments: A) Migrate more slowly and stay near the well B) Migrate faster and travel farther from the well C) Do not enter the gel at all D) Migrate at exactly the same speed regardless of size.
Correct answer: B — Migrate faster and travel farther from the well.
Reference: VU handout — Agarose gel electrophoresis.

2. Finalterm Solved MCQs

Q1. Real-time (quantitative) PCR differs from conventional PCR in that it: A) Uses no primers B) Measures the amount of DNA product as it is made, using fluorescence C) Works only on RNA D) Needs no DNA polymerase.
Correct answer: B — Measures the amount of DNA product as it is made, using fluorescence.
Reference: VU handout — Real-time PCR amplification.

Q2. The significance of the "heat shock" step in bacterial transformation is to: A) Kill the bacteria B) Facilitate the uptake of plasmid DNA into competent cells C) Cut the plasmid DNA D) Inactivate the antibiotic-resistance gene.
Correct answer: B — Facilitate the uptake of plasmid DNA into competent cells.
Reference: VU handout — Transformation of bacteria with plasmid DNA.

Q3. In blue-white screening, a white bacterial colony indicates that: A) The plasmid was lost B) The lacZ gene is intact and no insert is present C) The lacZα gene has been disrupted by an inserted DNA fragment (a recombinant plasmid) D) The bacteria are dead.
Correct answer: C — The lacZα gene has been disrupted by an inserted DNA fragment (a recombinant plasmid).
Reference: VU handout — Cloning; screening of recombinants.

Q4. Pure DNA has an absorbance ratio A260/A280 of about: A) 0.5 B) 1.0 C) 1.8 D) 2.5.
Correct answer: C — 1.8.
Reference: VU handout — Quantification of nucleic acids.

Q5. The CTAB method is used for the extraction of DNA from: A) Mammalian blood only B) Plant tissue C) Viruses only D) Purified proteins.
Correct answer: B — Plant tissue.
Reference: VU handout — DNA extraction from plant tissue: CTAB.

Q6. SDS-PAGE separates proteins mainly on the basis of their: A) Charge only B) Molecular weight (size) C) Shape only D) Solubility in water.
Correct answer: B — Molecular weight (size).
Reference: VU handout — Analysis of proteins by SDS-PAGE.

Q7. Restriction fragment length polymorphism (RFLP) detects a mutation because: A) The mutation changes the length of the whole chromosome B) The mutation creates or destroys a restriction site, changing the sizes of the fragments produced C) The mutation prevents PCR from working D) The mutation changes the colour of the DNA.
Correct answer: B — The mutation creates or destroys a restriction site, changing the sizes of the fragments produced.
Reference: VU handout — Detection of mutations by restriction fragment length polymorphism.

Q8. Synthesis of cDNA from an mRNA template requires the enzyme: A) DNA ligase B) Reverse transcriptase C) Taq polymerase D) Restriction endonuclease.
Correct answer: B — Reverse transcriptase.
Reference: VU handout — cDNA synthesis (RT-PCR).

Q9. Nested PCR increases the specificity of amplification because it: A) Uses a higher denaturation temperature B) Uses two successive pairs of primers, the second pair binding inside the first product C) Uses no primers at all D) Amplifies the whole genome.
Correct answer: B — Uses two successive pairs of primers, the second pair binding inside the first product.
Reference: VU handout — Polymerase chain reaction and its types.

Q10. In the organic (phenol-chloroform) method of DNA extraction, the purpose of phenol-chloroform is to: A) Break the DNA into small pieces B) Remove proteins from the DNA preparation C) Stain the DNA so it can be seen D) Copy the DNA.
Correct answer: B — Remove proteins from the DNA preparation.
Reference: VU handout — Isolation of DNA by organic method.

3. Midterm Subjective

Q1. Describe agarose gel electrophoresis: its principle, procedure, and the factors that affect how DNA fragments migrate.
Answer: Agarose gel electrophoresis separates DNA fragments by size. Principle: DNA is negatively charged (because of its phosphate groups), so when an electric current is applied across a gel, DNA fragments move towards the positive electrode (anode). The agarose gel acts as a molecular sieve: smaller fragments pass through the pores easily and travel farther, while larger fragments are held back. Procedure: (1) agarose powder is dissolved in buffer by heating and poured into a tray with a comb to form wells; (2) DNA samples mixed with loading dye are loaded into the wells, along with a DNA ladder (size marker); (3) an electric current is applied (typically 80-120 V) for 30-60 minutes; (4) the gel is stained with ethidium bromide (or a safer dye) and viewed under UV light, where the DNA appears as fluorescent bands. Factors affecting migration: size of the DNA fragment (the main factor — smaller moves faster); agarose concentration (higher concentration gives smaller pores and better resolution of small fragments); voltage (higher voltage moves DNA faster but can blur bands and overheat the gel); the conformation of the DNA (supercoiled DNA moves faster than linear DNA of the same size); and the buffer and running time. The technique is used to check the size and quality of DNA after extraction, after restriction digestion, and after PCR.
Reference: VU handout — Agarose gel electrophoresis.

Q2. Compare conventional PCR, reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR): what is the starting material, what is measured, and when is each used?
Answer: All three use repeated cycles of denaturation (about 94 °C), annealing (about 50-65 °C) and extension (about 72 °C) with a heat-stable DNA polymerase, but they differ in purpose. (1) Conventional (endpoint) PCR starts from DNA and amplifies a specific target sequence; the product is checked at the end, usually by gel electrophoresis. It is used to detect the presence of a DNA sequence, to prepare DNA for cloning or sequencing, and in DNA fingerprinting. It tells you whether the target is present, not how much was there. (2) RT-PCR starts from RNA: reverse transcriptase first copies the RNA into complementary DNA (cDNA), which is then amplified by PCR. It is used to detect gene expression — whether a particular gene is switched on in a tissue — and to work with RNA viruses. (3) Real-time quantitative PCR (qPCR) starts from DNA (or cDNA, as RT-qPCR) and measures the product as it is made, using a fluorescent dye or probe; the cycle at which fluorescence crosses a threshold (Ct value) is proportional to the starting amount. It is therefore used when the amount matters: measuring viral load, quantifying gene-expression levels, and detecting small amounts of a pathogen. In short: conventional PCR asks "is it there?", RT-PCR asks "is this gene expressed?", and qPCR asks "how much is there?".
Reference: VU handout — Polymerase chain reaction and its types; cDNA synthesis (RT-PCR); quantitative real-time PCR.

Q3. What are restriction enzymes? Explain sticky ends and blunt ends, and describe the uses of restriction enzymes in cloning.
Answer: Restriction enzymes (restriction endonucleases) are enzymes, mostly from bacteria, that cut DNA at specific recognition sequences, usually 4-8 base pairs long and usually palindromic (the sequence reads the same on both strands). They are the bacterium's defence against invading viruses: they cut foreign DNA while the bacterium's own DNA is protected by methylation. When an enzyme cuts the two strands at different positions (staggered cut), it leaves short single-stranded overhangs called sticky ends (e.g. EcoRI leaves 5'-AATT overhangs); these can base-pair with a complementary sticky end from another fragment cut by the same enzyme, making it easy to join the two pieces. When an enzyme cuts both strands at the same position, it leaves blunt ends with no overhang; blunt ends can be joined to any other blunt end but ligate less efficiently. Uses in cloning: to cut the vector and the donor DNA with the same enzyme so the fragments have matching ends; to cut out a gene of interest from genomic DNA; to prepare restriction maps of DNA; and, in RFLP analysis, to reveal mutations that create or destroy a restriction site. Without restriction enzymes, recombinant DNA technology would not be possible.
Reference: VU handout — Restriction digestion of DNA and preparation of restriction maps.

Q4. Describe the isolation of DNA from human blood by the organic (phenol-chloroform) method: the steps and the purpose of each step.
Answer: The organic method isolates DNA from white blood cells in a blood sample. Steps: (1) Lysis — blood cells are broken open with a lysis buffer containing detergent (SDS), which disrupts the cell and nuclear membranes; proteinase K is added to digest proteins, including the histones that package DNA and the nucleases that would degrade it. (2) Phenol-chloroform extraction — an equal volume of phenol:chloroform is added and mixed; phenol denatures the remaining proteins, which collect at the interface (or in the organic phase) when the tube is centrifuged, while the DNA stays in the upper aqueous phase. The aqueous phase is carefully removed to a new tube, and the extraction may be repeated for purity. (3) Precipitation — salt (e.g. sodium acetate) and cold ethanol or isopropanol are added; DNA is insoluble in alcohol and precipitates as a white, thread-like mass that can be spooled out or pelleted by centrifugation. (4) Washing — the pellet is washed with 70% ethanol to remove salts, air-dried, and dissolved in TE buffer or water. The quality of the DNA is then checked by gel electrophoresis and by spectrophotometry (A260/A280 about 1.8 for pure DNA). The method gives high-quality, high-molecular-weight DNA suitable for PCR, cloning and sequencing, but phenol is toxic and must be handled in a fume hood with gloves.
Reference: VU handout — Isolation of DNA by organic method (parts A and B); isolation of nucleic acids.

Q5. What is reverse transcription, and why is cDNA needed? Explain how cDNA is made and where it is used.
Answer: Reverse transcription is the synthesis of DNA from an RNA template by the enzyme reverse transcriptase (first found in retroviruses). The product is called complementary DNA (cDNA). cDNA is needed for two main reasons: (1) Many genes of interest are studied through their messenger RNA (mRNA), which shows which genes are actually expressed in a cell; but RNA is fragile and cannot be cloned into a plasmid or amplified by ordinary PCR, so it must first be converted into stable DNA. (2) Eukaryotic genes contain introns, which are removed when mRNA is made; cDNA therefore contains only the coding sequence (exons), so it can be expressed directly in bacteria, which cannot splice introns. How cDNA is made: mRNA is purified (often using its poly-A tail), a primer (oligo-dT, which binds the poly-A tail, or random hexamers) is annealed, and reverse transcriptase extends the primer to make a DNA-RNA hybrid; the RNA strand is then removed (by RNase H) and a second DNA strand is synthesised, giving double-stranded cDNA. Uses: measuring gene expression (RT-PCR and RT-qPCR), building cDNA libraries, cloning eukaryotic genes for expression in bacteria, and studying RNA viruses.
Reference: VU handout — cDNA synthesis (RT-PCR); isolation of RNA.

4. Finalterm Subjective

Q1. Describe how competent bacterial cells are prepared and how transformation is carried out by heat shock and by electroporation.
Answer: Transformation is the uptake of foreign (plasmid) DNA by a bacterial cell. Natural E. coli cells take up DNA very poorly, so they must first be made competent. Chemical competence: cells in the log phase of growth are chilled and treated with ice-cold calcium chloride (CaCl2); the Ca2+ ions neutralise the negative charges on the DNA and the cell membrane and make the membrane more permeable, and the cells are kept cold to keep the membrane in a state that can take up DNA. Transformation by heat shock: plasmid DNA is mixed with the competent cells on ice, the tube is then given a brief heat shock (42 °C for 45-90 seconds), which creates a thermal imbalance that sweeps the DNA into the cell, and the cells are returned to ice; they are then allowed to recover in nutrient broth (e.g. SOC medium) at 37 °C for about an hour so that they can express the plasmid's antibiotic-resistance gene. Electroporation: instead of chemicals, a brief, high-voltage electric pulse (about 2.5 kV) is applied to a mixture of cells and DNA in a cuvette; the pulse makes temporary pores in the membrane through which the DNA enters. Electroporation is faster and often more efficient, but needs a specialised electroporator and very clean, salt-free DNA, whereas heat shock is cheaper and is the standard teaching method. Transformed cells are then selected by plating on agar containing the antibiotic to which the plasmid gives resistance — only cells that took up the plasmid grow.
Reference: VU handout — Preparation of competent cells; transformation of bacteria with plasmid DNA.

Q2. Compare Southern blotting, Northern blotting and Western blotting: what molecule does each detect, and how is each carried out?
Answer: All three are blotting techniques in which molecules are separated (usually by gel electrophoresis) and transferred ("blotted") onto a membrane, where a labelled probe detects a specific target. (1) Southern blotting detects a specific DNA sequence: DNA is cut with restriction enzymes, separated on an agarose gel, denatured to single strands, transferred to a membrane, and hybridised with a labelled DNA probe complementary to the target; it is used for DNA fingerprinting, detecting gene deletions or mutations, and RFLP analysis. (2) Northern blotting detects a specific RNA: RNA is separated on a gel, transferred to a membrane, and hybridised with a labelled DNA or RNA probe; it is used to measure the expression of a particular gene (how much mRNA is present in a tissue). (3) Western blotting detects a specific protein: proteins are separated by SDS-PAGE, transferred to a membrane, and detected with a specific antibody (primary antibody against the target, then a labelled secondary antibody); it is used to confirm that a protein is present, to estimate its size and amount, and in diagnostics (e.g. confirmatory testing for HIV). The names are a joke on the history: Southern is named after Edwin Southern, its inventor, while Northern and Western were named by analogy. All three rely on specific binding — base-pairing for Southern and Northern, antibody binding for Western — to pick one molecule out of thousands.
Reference: VU handout — Blotting techniques; detection of nucleic acids and proteins.

Q3. Describe restriction fragment length polymorphism (RFLP): its principle, procedure and applications.
Answer: RFLP is a technique that detects differences in DNA sequence by looking at the pattern of fragments produced when DNA is cut with restriction enzymes. Principle: a mutation (a single base change, insertion or deletion) can create a new restriction site or destroy an existing one, so the same region of DNA is cut into fragments of different lengths in different individuals. Procedure: (1) DNA is extracted from the sample; (2) it is digested with a restriction enzyme; (3) the fragments are separated by agarose gel electrophoresis; (4) the fragments are transferred to a membrane (Southern blot) and hybridised with a labelled probe for the region of interest, revealing a pattern of bands. If two samples give different band patterns, their DNA differs at a restriction site in that region. Applications: detecting mutations that cause genetic diseases (e.g. sickle-cell anaemia, where the mutation destroys a restriction site); DNA fingerprinting and paternity testing; forensic identification; and genetic mapping, because RFLP markers can be followed through families. RFLP was one of the first DNA-based markers used in genetics; today it has largely been replaced by PCR-based methods (e.g. STR analysis and sequencing), which need much less DNA, but the principle — that sequence differences change restriction patterns — is still the basis of many diagnostic tests.
Reference: VU handout — Detection of mutations by restriction fragment length polymorphism; DNA fingerprinting.

Q4. Describe the methods used to quantify nucleic acids: spectrophotometry (A260, A260/A280), gel electrophoresis and fluorometry. What does each tell you?
Answer: Three methods are commonly used, and they give different information. (1) UV spectrophotometry: nucleic acids absorb UV light most strongly at 260 nm. The absorbance at 260 nm (A260) is used to calculate concentration — an A260 of 1.0 corresponds to about 50 µg/mL of double-stranded DNA (40 µg/mL for RNA). The ratio A260/A280 measures purity: pure DNA gives about 1.8 and pure RNA about 2.0; a lower ratio indicates protein contamination (proteins absorb at 280 nm), while a low A260/A230 indicates contamination with salts or organic solvents. Spectrophotometry is quick but cannot distinguish intact DNA from degraded DNA, and it measures all nucleic acids together. (2) Agarose gel electrophoresis: a known amount of DNA is run beside standards of known concentration, and the brightness of the bands is compared; the gel also shows whether the DNA is intact (a sharp band) or degraded (a smear), and whether RNA or protein contamination is present. (3) Fluorometry: fluorescent dyes that bind specifically to DNA (e.g. PicoGreen) or RNA are used, and the fluorescence is measured in a fluorometer (e.g. Qubit); this is the most sensitive and specific method, because the dye binds only double-stranded DNA, so it is not confused by free nucleotides, RNA or protein. In practice, a laboratory checks concentration by spectrophotometry, checks integrity on a gel, and uses fluorometry when accurate, low-level quantification is needed (e.g. before sequencing).
Reference: VU handout — Quantification of nucleic acid; quantification of DNA by gel electrophoresis; quantification of RNA by Nanodrop.

Q5. Describe SDS-PAGE: its principle, procedure, and what it is used for.
Answer: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separates proteins by their molecular weight. Principle: the detergent SDS denatures proteins and coats them with a uniform negative charge, so that the charge-to-mass ratio is the same for all proteins; the proteins then separate in the polyacrylamide gel only by size — smaller proteins pass through the pores more easily and travel farther. A reducing agent (e.g. beta-mercaptoethanol) is usually added to break disulfide bonds, so each protein runs as a single unfolded chain. Procedure: (1) protein samples are mixed with SDS sample buffer and heated to denature them; (2) samples are loaded into wells of a polyacrylamide gel alongside molecular-weight markers; (3) an electric current is applied and the proteins migrate towards the positive electrode; (4) the gel is stained (e.g. with Coomassie blue) to reveal the protein bands, or the proteins are transferred to a membrane for Western blotting. The size of an unknown protein is estimated by comparing how far it travelled with the marker proteins. Uses: checking the purity of a protein preparation, estimating the molecular weight of a protein, comparing protein samples (e.g. before and after induction of expression), and as the first step of Western blotting. Unlike agarose gel electrophoresis of DNA, SDS-PAGE separates molecules by size after their charge has been made uniform, rather than by their natural charge.
Reference: VU handout — Analysis of proteins by SDS-PAGE.

FAQ

Q: Is the Midterm from Lectures 1-22? A: Yes — Midterm covers Lectures 1-22 and Finalterm covers Lectures 23-45.

Q: Does the quiz work from this post? A: Yes — use the 'Take the quiz' link above; the quiz is already live at ?code=BIO203 with Midterm 10 and Finalterm 10.

Q: Are the answers verified? A: Yes — all 30 items are marked Verified, solved independently and cross-checked against at least two independent sources.

More Solved Papers

See the Solved MCQs page and the midterm and finalterm mega hubs for solved papers for every subject.