DNA Extraction
1. In organic liquid phase extraction using phenol and chloroform, in which layer is the DNA located after the sample is centrifuged?
A. The interface between layers
B. The solid pellet at the very bottom
C. The top aqueous layer
D. The bottom organic layer
C. The top aqueous layer
Centrifugation separates the mixture into layers based on density and solubility, with DNA remaining in the upper water-based phase.
6. What is the specific role of Mg2+ ions in a standard Polymerase Chain Reaction (PCR)?
A. It lowers the melting temperature of the DNA double helix
B. It provides the template sequence for amplification
C. It acts as a cofactor for the DNA polymerase enzyme
D. It serves as the building block for the new DNA strand
C. It acts as a cofactor for the DNA polymerase enzyme
Magnesium ions are essential for the catalytic activity of thermostable DNA polymerases like Taq during the synthesis of new DNA strands.
11. Which technique is primarily used to visualize whole chromosomes to detect large-scale genetic abnormalities such as trisomies?
A. Karyotyping
B. ELISA
C. Real-Time PCR
D. Western Blotting
A. Karyotyping
Karyotyping involves staining and imaging the full set of chromosomes to identify numerical or large structural changes.
16. Which of the following is a disadvantage of ELISA when compared to PCR-based molecular testing for infectious diseases?
A. It requires more advanced and expensive equipment
B. It is unable to provide any quantitative data
C. It can only detect nucleic acids and not proteins
D. It typically detects infection at a later stage
D. It typically detects infection at a later stage
ELISA often relies on detecting the body's immune response (antibodies), which takes time to develop after the initial infection.
21. In Western Blotting, what is the purpose of adding Sodium Dodecyl Sulfate (SDS) to the protein sample?
A. To unfold proteins and give them a uniform net negative charge
B. To act as a primary antibody for target detection
C. To catalyze the chemiluminescent reaction for imaging
D. To block unoccupied sites on the nitrocellulose membrane
A. To unfold proteins and give them a uniform net negative charge
SDS is an anionic detergent that denatures proteins and ensures they are separated based solely on their molecular weight during electrophoresis.
2. Which of the following is a common storage condition for isolated DNA to ensure long-term stability?
A. Stored in 2-mercaptoethanol at room temperature
B. Stored at 37°C in distilled water
C. Stored in a phenol:chloroform mixture
D. Stored at -20°C in TE buffer
D. Stored at - 20°C in TE buffer
Freezing at -20°C slows enzymatic activity, and TE buffer helps maintain a stable pH and chelates ions that could damage DNA.
7. In Real-Time PCR (qPCR) data analysis, what does an earlier Cycle Threshold (Ct) value indicate about the sample?
A. The sample had a higher initial quantity of target DNA
B. The PCR reaction was inefficient or failed
C. The sample contains a high concentration of inhibitors
D. The DNA polymerase was denatured too early
A. The sample had a higher initial quantity of target DNA
An earlier Ct means the fluorescence signal crossed the detection threshold in fewer cycles, which occurs when more template is present at the start.
12. During agarose gel electrophoresis, toward which electrode does the DNA migrate, and what property of DNA causes this movement?
A. Positive electrode (anode) because of the hydrophobic nature of the nitrogenous bases.
B. Negative electrode (cathode) because the DNA is denatured by the buffer.
C. Positive electrode (anode) because of the negative charge on the phosphate backbone.
D. Negative electrode (cathode) because DNA is positively charged.
C. Positive electrode (anode) because of the negative charge on the phosphate backbone.
DNA is negatively charged and is therefore attracted to the positive electrode during the application of an electric current.
17. How is the precise concentration of an analyte determined in a Quantitative ELISA?
A. By calculating the ratio of phenol to chloroform used in the buffer
B. By visually checking if the color is darker than the negative control
C. By comparing the sample signal to a standard curve of known concentrations
D. By measuring the cycle threshold (Ct) on a thermal cycler
C. By comparing the sample signal to a standard curve of known concentrations
A standard curve created from serial dilutions allows the interpolation of unknown sample concentrations based on their signal intensity.
22. During the SDS-PAGE step of a Western Blot, what determines the rate at which proteins migrate toward the positive electrode?
A. Molecular weight (size)
B. The amount of primary antibody added
C. The sequence of the amino acids
D. The pH of the storage buffer
A. Molecular weight (size)
The polyacrylamide gel acts as a molecular sieve; smaller proteins navigate the matrix faster than larger ones.
3. A researcher measures the absorbance of a DNA sample at 260 nm and obtains a value of 0.220. If the sample was diluted 1:10 before measurement, what is the final DNA concentration?
A. 110 μg/mL
B. 44 μg/mL
C. 11 μg/mL
D. 1100 μg/mL
A. 110 ug/mL
Using the formula D = (A260) × 50 ug/mL x dilution factor, the calculation is 0.220 × 50 × 10 = 110.
8. In the context of mericon real-time PCR assays, what is the function of the 'Internal Control' (IC) included in the primer/probe mix?
A. To detect the presence of PCR inhibitors and verify the success of the reaction.
B. To act as a quencher for the primary target's fluorophore.
C. To increase the sensitivity of the assay to detect fewer than 10 copies.
D. To bind non-specifically to all double-stranded DNA produced.
A. To detect the presence of PCR inhibitors and verify the success of the reaction.
The internal control ensures that a negative result is truly due to the absence of target DNA rather than a failure of the PCR process itself.
13. What is the fundamental principle behind Fluorescence In Situ Hybridization (FISH)?
A. Proteins are separated by size using an electric current
B. Fluorescently labeled probes bind to specific complementary DNA or RNA sequences
C. Enzymes convert a colorless substrate into a colored product
D. Radioactive isotopes are used to measure DNA synthesis rates
B. Fluorescently labeled probes bind to specific complementary DNA or RNA sequences
FISH relies on the ability of a single-stranded probe to find and hybridize with its matching sequence within a fixed cell or tissue sample.
18. Which enzymatic activity of reverse transcriptase is essential for the degradation of the RNA strand within an RNA:DNA hybrid during the synthesis of double-stranded cDNA?
A. RNA-dependent DNA polymerase
B. DNA-dependent DNA polymerase
C. DNA-dependent RNA polymerase
D. RNase H
D. RNase H
RNase H specifically degrades the RNA portion of a DNA:RNA hybrid, allowing for the synthesis of the second DNA strand.
23. In Western blotting, what is the primary purpose of using a high concentration of polyacrylamide in the 'running' gel?
A. To increase the net negative charge on all proteins in the sample.
B. To provide a surface for the antibodies to bind directly to the proteins.
C. To resolve and separate lower molecular weight proteins.
D. To allow large proteins to migrate faster through the matrix.
C. To resolve and separate lower molecular weight proteins.
Higher acrylamide concentrations create a denser 'maze,' which is necessary to differentiate smaller proteins by size.
4. During a DNA purity check, a sample yields an (A260−A320)/(A280−A320) ratio of 1.4. What is the most likely interpretation of this result?
A. The sample is highly pure double-stranded DNA.
B. The DNA has been successfully degraded into single strands.
C. The concentration of DNA is too high for accurate measurement.
D. The sample contains significant protein or phenol contamination.
D. The sample contains significant protein or phenol contamination.
A ratio below 1.8 indicates that contaminants such as proteins or organic solvents are present in the extract.
9. Which specific modification distinguishes Reverse Transcription PCR (RT-PCR) from conventional PCR?
A. The requirement for multiple cycles of heating and cooling.
B. The use of Phenol and Chloroform to stabilize the template.
C. The use of Reverse Transcriptase to convert RNA into complementary DNA (cDNA) before amplification.
D. The addition of fluorescent probes for real-time monitoring.
C. The use of Reverse Transcriptase to convert RNA into complementary DNA (cDNA) before amplification.
Conventional PCR requires a DNA template; RT-PCR adds a preliminary step to synthesize DNA from an RNA source.
14. Which chromosomal abnormality is correctly matched with its clinical description?
A. Fragile X Syndrome: A translocation between chromosomes 9 and 22.
B. Turner Syndrome: A female condition (45,X) leading to short stature and underdeveloped ovaries.
C. Down Syndrome: A monosomy involving chromosome 21.
D.Klinefelter Syndrome: A female condition caused by the absence of one X chromosome.
B. Turner Syndrome: A female condition (45, X) leading to short stature and underdeveloped ovaries.
Turner syndrome is characterized by the complete or partial absence of one X chromosome in females, resulting in specific physical and reproductive traits.
19. Which component of an antibody monomer determines its biological properties and classification into specific isotypes such as IgG or IgA?
A. The variable region
B. The light chain
C. The Fc fragment
D. The Fab fragment
C. The Fc fragment
The Fc (fragment crystallizable) region, composed of the constant heavy chains, determines the antibody's class and effector functions.
24. What is the specific mechanistic role of 2−mercaptoethanol in Western Blot sample preparation?
A. It inhibits protease enzymes to prevent the degradation of the protein of interest.
B. It acts as a reducing agent to break covalent disulfide bonds between cysteine residues.
C. It facilitates the transfer of proteins from the polyacrylamide gel to the nitrocellulose membrane.
D. It coats the protein in a uniform negative charge to allow for separation by size.
B. It acts as a reducing agent to break covalent disulfide bonds between cysteine residues.
Complete denaturation of proteins often requires breaking strong disulfide crosslinks, which is achieved using reducing agents like 2 - mercaptoethanol.
5. Which component of inorganic DNA extraction is responsible for the 'salting out' of proteins after cell lysis?
A. Isopropanol
B. EDTA
C. SDS
D. Sodium acetate
D. Sodium acetate
High concentrations of salts like sodium acetate reduce the solubility of proteins, causing them to precipitate so they can be removed by centrifugation.
10. In qPCR, how does a 'TaqMan' probe generate a fluorescent signal?
A. The 5′−3′ exonuclease activity of Taq polymerase cleaves the probe during extension.
B. The probe fluoresces only when it is bound to double-stranded DNA.
C. The probe undergoes a conformational change from a hairpin to a linear structure.
D. Two adjacent probes hybridize to bring a donor and acceptor fluorophore together.
A. The 5' - 3' exonuclease activity of Tag polymerase cleaves the probe during extension.
Cleavage separates the fluorophore from the quencher, which stops the suppression of the signal and allows detection.
15. In Q-banding, what characterizes the DNA regions that appear as 'quinacrine-intense' bright bands under a fluorescent microscope?
A. Areas specifically pretreated with trypsin
B. Regions primarily containing euchromatin
C. DNA rich in adenine and thymine
D. DNA rich in guanine and cytosine
C. DNA rich in adenine and thymine
Quinacrine mustard has a higher affinity for AT-rich regions, which results in bright fluorescence in heterochromatic areas.
20. Which priming strategy in reverse transcription is most likely to result in a lower cDNA yield due to its high sensitivity to the secondary structure of the RNA template?
A. Oligo (dT) primers
B. Random hexamer primers
C. Gene-specific primers
D. Sequence-independent primers
A. Oligo (dT) primers
Because they must bind to the 3' poly-A tail, any secondary structure blocking that end or the path of the polymerase can significantly reduce efficiency.
25. In the context of Western blotting, what is the primary advantage of using a PVDF membrane over a nitrocellulose membrane?
A. It is significantly less expensive for routine clinical use.
B. It has a lower affinity for proteins, reducing background noise.
C. It is more durable and can withstand repeated probings.
D. It does not require an electric current for protein transfer.
C. It is more durable and can withstand repeated probings.
The material specifies that nitrocellulose is fragile, whereas PV DF is more pliable and strong.