PCR & GE

*Question: Who invented PCR and in what year?
A) Dr. James Watson, 1953
B) Dr. Kary Mullis, 1983
C) Dr. Frederick Sanger, 1977
D) Dr. Linus Pauling, 1990

*Answer: B) Dr. Kary Mullis, 1983

*Question: What type of technique is PCR?
A) In-vivo amplification technique
B) In-vitro amplification technique
C) In-situ hybridization technique
D) Immunological detection technique

*Answer: B) In-vitro amplification technique

*Question: Which of the following is NOT a component of PCR?
A) Taq DNA polymerase
B) Magnesium Chloride
C) RNA polymerase
D) dNTPs

*Answer: C) RNA polymerase

*Question: What is the maximum template DNA size for PCR?
A) 1 Kb
B) 2 Kb
C) 3 Kb
D) 5 Kb

*Answer: C) 3 Kb

*Question: What is the recommended template DNA concentration in a 50 uL total reaction mixture?
A) 0.01–0.05 ug
B) 0.1–1 ug
C) 1–5 ug
D) 5–10 ug

*Answer: B) 0.1–1 ug

*Question: What is the ideal GC content for PCR primers?
A) 20–30%
B) 30–40%
C) 40–60%
D) 60–80%

*Answer: C) 40–60%

*Question: What is the recommended length of PCR primers?
A) 5–10 nucleotides
B) 10–15 nucleotides
C) 15–30 nucleotides
D) 30–50 nucleotides

*Answer: C) 15–30 nucleotides

*Question: What is the primer concentration used in a 50 uL PCR reaction?
A) 10 pmol (0.2 uM)
B) 25 pmol (0.5 uM)
C) 50 pmol (1 uM)
D) 100 pmol (2 uM)

*Answer: C) 50 pmol (1 uM)

*Question: The forward primer is complementary to the 3' end of which strand?
A) Sense strand (5'-3')
B) Antisense strand (3'-5')
C) Coding strand
D) mRNA strand

*Answer: B) Antisense strand (3'-5')

*Question: The reverse primer is also known as:
A) Upstream oligonucleotide
B) Downstream oligonucleotide
C) Sense oligonucleotide
D) Template oligonucleotide

*Answer: B) Downstream oligonucleotide

*Question: The forward primer is also known as:
A) Downstream oligonucleotide
B) Antisense oligonucleotide
C) Upstream oligonucleotide
D) Reverse oligonucleotide

*Answer: C) Upstream oligonucleotide

*Question: When the lecture says "1 primer," how many actual primers does that represent?
A) 1
B) 2
C) 3
D) 4

*Answer: B) 2

*Question: What is the KCl concentration in the PCR buffer?
A) 100 mM KCl
B) 200 mM KCl
C) 500 mM KCl
D) 1000 mM KCl

*Answer: C) 500 mM KCl

*Question: What is the Tris-HCl concentration in the PCR buffer?
A) 50 mM Tris-HCl (8.3)
B) 100 mM Tris-HCl (8.3)
C) 200 mM Tris-HCl (8.3)
D) 500 mM Tris-HCl (8.3)

*Answer: B) 100 mM Tris-HCl (8.3)

*Question: What is the role of Magnesium Chloride in PCR?
A) It provides the DNA template
B) It is an essential cofactor of DNA polymerase
C) It provides energy for primer binding
D) It degrades non-specific amplicons

*Answer: B) It is an essential cofactor of DNA polymerase

*Question: What happens if too much magnesium is added to the PCR reaction?
A) The enzyme will not work
B) Non-specific amplifications will occur
C) The primer will not anneal
D) Denaturation will fail

*Answer: B) Non-specific amplifications will occur

*Question: What is the working concentration range of magnesium in the PCR assay?
A) 0.1 to 1.5 uM
B) 0.5 to 3.5 uM
C) 1.0 to 5.0 uM
D) 2.0 to 6.0 uM

*Answer: B) 0.5 to 3.5 uM

*Question: Which DNA polymerase is derived from Thermus aquaticus?
A) Pfu polymerase
B) KOD DNA polymerase
C) Taq polymerase
D) T4 polymerase

*Answer: C) Taq polymerase

*Question: Which DNA polymerase is derived from Pyrococcus furiosus?
A) Taq polymerase
B) Pfu polymerase
C) KOD DNA polymerase
D) T7 polymerase

*Answer: B) Pfu polymerase

*Question: How many units of Taq DNA polymerase are used per 50 uL reaction?
A) 0.5 U
B) 1.0 U
C) 1.25 U
D) 2.0 U

*Answer: C) 1.25 U

*Question: At what pH and concentration are dNTPs stored?
A) 5 mM, pH 6.0
B) 10 mM, pH 7.0
C) 20 mM, pH 8.0
D) 50 mM, pH 7.5

*Answer: B) 10 mM, pH 7.0

*Question: What is the working concentration range of dNTPs added in the PCR assay?
A) 1–10 uM
B) 5–15 uM
C) 20–200 uM
D) 200–500 uM

*Answer: C) 20–200 uM

*Question: What are the four dNTPs used in PCR?
A) dATP, dGTP, dCTP, dUTP
B) dATP, dGTP, dCTP, dTTP
C) ATP, GTP, CTP, TTP
D) dAMP, dGMP, dCMP, dTMP

*Answer: B) dATP, dGTP, dCTP, dTTP

*Question: What is the mixture of all PCR components collectively called?
A) Elution buffer
B) PCR mix, mastermix, or cocktail
C) Loading dye
D) Hybridization solution

*Answer: B) PCR mix, mastermix, or cocktail

*Question: Which instrument is used to run the PCR thermal cycling process?
A) Centrifuge
B) Spectrophotometer
C) Thermal cycler
D) Electrophoresis chamber

*Answer: C) Thermal cycler

*Question: How many basic steps are included in each PCR cycle?
A) 2
B) 3
C) 4
D) 5

*Answer: B) 3

*Question: What are the three basic steps of each PCR cycle in correct order?
A) Annealing, Denaturation, Extension
B) Extension, Annealing, Denaturation
C) Denaturation, Annealing, Extension
D) Denaturation, Extension, Annealing

*Answer: C) Denaturation, Annealing, Extension

*Question: What is the temperature range for the denaturation step in PCR?
A) 50–60°C
B) 68–75°C
C) 90–96°C
D) 37–42°C

*Answer: C) 90–96°C

*Question: What is the time range for the denaturation step?
A) 10–20 sec
B) 20–60 sec
C) 60–90 sec
D) 90–120 sec

*Answer: B) 20–60 sec

*Question: What is the goal of the denaturation step?
A) Allow primers to bind to the template
B) Synthesize new DNA strands
C) Separate double-stranded DNA into single strands by melting hydrogen bonds
D) Activate the DNA polymerase

*Answer: C) Separate double-stranded DNA into single strands by melting hydrogen bonds

*Question: What is the temperature range for the annealing step?
A) 30–45°C
B) 50–70°C
C) 72–80°C
D) 90–96°C

*Answer: B) 50–70°C

*Question: What is the time range for the annealing step?
A) 5–10 sec
B) 10–15 sec
C) 20–90 sec
D) 90–120 sec

*Answer: C) 20–90 sec

*Question: Which step of PCR is considered the most critical?
A) Denaturation
B) Annealing
C) Extension
D) Initial denaturation

*Answer: B) Annealing

*Question: What is the goal of the annealing step?
A) Melt the hydrogen bonds of dsDNA
B) Ensure the primer binds/anneals to the DNA template
C) Extend the primer using dNTPs
D) Denature the DNA polymerase

*Answer: B) Ensure the primer binds/anneals to the DNA template

*Question: What is the temperature range for the extension step?
A) 50–60°C
B) 60–65°C
C) 68–75°C
D) 90–96°C

*Answer: C) 68–75°C

*Question: What is the time range for the extension step?
A) 5–10 sec
B) 10–60 sec
C) 60–90 sec
D) 90–180 sec

*Answer: B) 10–60 sec

*Question: What is the maximum temperature for the extension process?
A) 68°C
B) 72°C
C) 75°C
D) 80°C

*Answer: C) 75°C

*Question: What is the standard number of cycles in one PCR run?
A) 10–15 cycles
B) 20–25 cycles
C) 30–35 cycles
D) 40–45 cycles

*Answer: C) 30–35 cycles

*Question: After 35 cycles, approximately how many copies of DNA are produced?
A) 35 copies
B) 1 million copies
C) 68 billion copies
D) 100 trillion copies

*Answer: C) 68 billion copies

*Question: What type of amplification occurs in PCR after 30–35 cycles?
A) Linear amplification
B) Logarithmic amplification
C) Exponential amplification
D) Arithmetic amplification

*Answer: C) Exponential amplification

*Question: What is the major difference between conventional PCR and real-time PCR?
A) Real-time PCR uses Taq polymerase; conventional does not
B) Real-time PCR uses fluorescent dyes to monitor amplification during cycling
C) Conventional PCR requires RNA template; real-time uses DNA
D) Conventional PCR is faster than real-time PCR

*Answer: B) Real-time PCR uses fluorescent dyes to monitor amplification during cycling

*Question: What does CT value stand for in real-time PCR?
A) Cycle Time value
B) Cycle Threshold value
C) Critical Temperature value
D) Copy Total value

*Answer: B) Cycle Threshold value

*Question: What does a LOW CT value indicate?
A) Low initial DNA concentration
B) High initial DNA concentration
C) Failed amplification
D) Contamination

*Answer: B) High initial DNA concentration

*Question: What does a HIGH CT value indicate?
A) High initial DNA concentration
B) Successful amplification
C) Low initial DNA concentration
D) Non-specific amplification

*Answer: C) Low initial DNA concentration

*Question: What cycle range is considered "early" amplification in real-time PCR?
A) 5–10 cycles
B) 15–20 cycles
C) 25–30 cycles
D) 35–40 cycles

*Answer: B) 15–20 cycles

*Question: What cycle range is considered "late" amplification in real-time PCR?
A) 5–15 cycles
B) 10–20 cycles
C) Greater than 20 cycles
D) Greater than 35 cycles

*Answer: C) Greater than 20 cycles

*Question: In a real-time PCR amplification plot, which sample has the highest initial DNA concentration?
A) The sample with the highest CT value
B) The sample with the lowest CT value
C) The sample that amplifies last
D) The negative control

*Answer: B) The sample with the lowest CT value

*Question: What does RT-PCR stand for?
A) Real-Time Polymerase Chain Reaction
B) Reverse Transcription PCR
C) Rapid Thermal PCR
D) Recombinant Template PCR

*Answer: B) Reverse Transcription PCR

*Question: What enzyme does RT-PCR use to convert RNA to DNA?
A) DNA polymerase
B) RNA polymerase
C) Reverse transcriptase
D) Helicase

*Answer: C) Reverse transcriptase

*Question: What does RT-PCR produce first before conventional PCR techniques are applied?
A) mRNA
B) Complementary DNA (cDNA)
C) Plasmid DNA
D) Genomic DNA

*Answer: B) Complementary DNA (cDNA)

*Question: What is an advantage of Multiplex PCR?
A) Requires only one primer
B) Can amplify multiple targets simultaneously in a single PCR reaction
C) Does not require a thermal cycler
D) Uses RNA as the template

*Answer: B) Can amplify multiple targets simultaneously in a single PCR reaction

*Question: What is a disadvantage of conventional PCR?
A) Cannot amplify large DNA fragments
B) Requires post-PCR analysis (e.g., gel electrophoresis) for detection
C) Cannot be used for cloning
D) Uses fluorescent dyes that are expensive

*Answer: B) Requires post-PCR analysis (e.g., gel electrophoresis) for detection

*Question: What is gel electrophoresis performed after in the molecular biology workflow?
A) Sample collection
B) Nucleic acid extraction
C) Amplification using PCR
D) Quality checking

*Answer: C) Amplification using PCR

*Question: What is the principle of electrophoresis?
A) Separation of molecules based on their temperature
B) Charged molecules migrate in response to an electrical field
C) Molecules are separated by size using centrifugal force
D) Molecules are separated by their solubility

*Answer: B) Charged molecules migrate in response to an electrical field

*Question: Which of the following is NOT a factor affecting the rate of migration in gel electrophoresis?
A) Strength of the field (voltage)
B) Net charge, size, and shape of the molecules
C) Color of the loading dye
D) Agarose concentration

*Answer: C) Color of the loading dye

*Question: What is the standard voltage used in gel electrophoresis?
A) 50 volts
B) 80 volts
C) 100 volts
D) 150 volts

*Answer: C) 100 volts

*Question: What can happen if the voltage is too high in gel electrophoresis?
A) DNA will not migrate
B) It can generate heat and distort the gel, affecting resolution
C) The buffer will evaporate
D) The agarose will dissolve

*Answer: B) It can generate heat and distort the gel, affecting resolution

*Question: Why is the buffer important in gel electrophoresis?
A) It colors the DNA bands
B) It provides ions for even distribution of electricity
C) It denatures the DNA before migration
D) It acts as a lubricant for the gel

*Answer: B) It provides ions for even distribution of electricity

*Question: What happens if the buffer has insufficient ions?
A) DNA migrates too fast
B) Non-specific bands appear
C) There will be no conduction of electricity and no migration
D) The gel will dissolve

*Answer: C) There will be no conduction of electricity and no migration

*Question: What is the stock concentration of Tris-Borate-EDTA (TBE) buffer?
A) 5x
B) 10x
C) 50x
D) 100x

*Answer: B) 10x

*Question: What is the stock concentration of Tris-Acetate-EDTA (TAE) buffer?
A) 5x
B) 10x
C) 50x
D) 100x

*Answer: C) 50x

*Question: In the school laboratory (SPC), what concentration of buffer is used?
A) 0.1x
B) 0.25x
C) 0.5x
D) 1x

*Answer: B) 0.25x

*Question: What is the working solution concentration range for TAE buffer?
A) 0.1–0.5x
B) 0.5–1.0x
C) 1.0–2.0x
D) 2.0–5.0x

*Answer: B) 0.5–1.0x

*Question: Which buffer has stronger buffering capacity — TAE or TBE?
A) TAE
B) TBE
C) They are equal
D) Depends on the DNA size

*Answer: B) TBE

*Question: In gel electrophoresis, DNA separation is based on:
A) Charge
B) Color
C) Size
D) Temperature

*Answer: C) Size

*Question: In gel electrophoresis, DNA migration is based on:
A) Size
B) Charge
C) Shape only
D) Molecular weight only

*Answer: B) Charge

*Question: Since DNA molecules are negatively charged (anions), they migrate toward which electrode?
A) Cathode (negative electrode)
B) Anode (positive electrode)
C) They do not migrate
D) Both electrodes equally

*Answer: B) Anode (positive electrode)

*Question: A smaller DNA fragment in gel electrophoresis will:
A) Migrate slower and stay near the well
B) Migrate faster and travel farther from the well
C) Not migrate at all
D) Migrate to the negative electrode

*Answer: B) Migrate faster and travel farther from the well

*Question: A larger DNA fragment in gel electrophoresis will:
A) Migrate faster and travel farther
B) Migrate slower and stay near the well
C) Migrate to the cathode
D) Dissolve in the buffer

*Answer: B) Migrate slower and stay near the well

*Question: A higher agarose concentration creates:
A) Larger pores
B) Smaller pores
C) No change in pore size
D) Dissolved gel

*Answer: B) Smaller pores

*Question: A lower agarose concentration creates:
A) Smaller pores
B) Larger pores
C) No pores
D) Uniform pore size

*Answer: B) Larger pores

*Question: What is the standard agarose concentration used as a "safety net" for gel electrophoresis?
A) 0.5%
B) 0.8%
C) 1%
D) 1.5%

*Answer: C) 1%

*Question: What agarose concentration range is appropriate for separating DNA fragments of 250bp to 12 kb?
A) 0.5%
B) 0.8%
C) 1%
D) 1.2%

*Answer: C) 1%

*Question: What agarose concentration is used for DNA fragments ranging from 700bp to 25 kb?
A) 0.5%
B) 0.8%
C) 1%
D) 1.5%

*Answer: A) 0.5%

*Question: What agarose concentration is used for DNA fragments ranging from 80bp to 4 kb?
A) 0.5%
B) 1%
C) 1.2%
D) 1.5%

*Answer: D) 1.5%

*Question: Agarose is derived from which substance?
A) Polyacrylamide
B) Starch
C) Agar
D) Cellulose

*Answer: C) Agar

*Question: At approximately what temperature does agarose gel solidify?
A) 20°C
B) 40°C
C) 60°C
D) 80°C

*Answer: B) 40°C

*Question: What type of bonds hold agarose gel together?
A) Covalent and ionic bonds
B) Disulfide bonds
C) Weak hydrogen and hydrophilic bonds (hydrocolloids)
D) Peptide bonds

*Answer: C) Weak hydrogen and hydrophilic bonds (hydrocolloids)

*Question: What stain is commonly used to visualize DNA in gel electrophoresis?
A) Coomassie Blue
B) Crystal Violet
C) Ethidium Bromide (EtBr)
D) Silver stain

*Answer: C) Ethidium Bromide (EtBr)

*Question: How does Ethidium Bromide (EtBr) interact with DNA?
A) It binds to the phosphate backbone
B) It intercalates into the planar structure of DNA
C) It covalently bonds to the nitrogenous bases
D) It coats the sugar-phosphate backbone

*Answer: B) It intercalates into the planar structure of DNA

*Question: What color does EtBr-stained DNA emit under UV light?
A) Blue-green
B) Yellow
C) Red-orange (at 590 nm)
D) Purple

*Answer: C) Red-orange (at 590 nm)

*Question: UV absorbed by DNA is at:
A) 100 nm
B) 160 nm
C) 254 nm
D) 590 nm

*Answer: B) 160 nm

*Question: What is an alternative stain to EtBr mentioned in the lecture?
A) SYBR Green
B) Gel Red
C) Coomassie Blue
D) Acridine Orange

*Answer: B) Gel Red

*Question: What is the orientation of agarose gel electrophoresis?
A) Vertical
B) Diagonal
C) Horizontal
D) Circular

*Answer: C) Horizontal

*Question: What is the orientation of polyacrylamide gel electrophoresis (PAGE)?
A) Horizontal
B) Vertical
C) Circular
D) Diagonal

*Answer: B) Vertical

*Question: Which gel type is better for separating small DNA molecules?
A) Agarose gel
B) Starch gel
C) Polyacrylamide gel
D) Both equally

*Answer: C) Polyacrylamide gel

*Question: Which gel type is primarily used for DNA in gel electrophoresis (as described in the lecture)?
A) Polyacrylamide gel
B) Starch gel
C) Agarose gel
D) Silica gel

*Answer: C) Agarose gel

*Question: Can polyacrylamide gel be used to separate proteins?
A) No, only DNA
B) Yes, DNA or proteins depending on gel type
C) Only RNA
D) Only lipids

*Answer: B) Yes, DNA or proteins depending on gel type

*Question: What is a "ladder" in gel electrophoresis?
A) A type of buffer
B) A DNA marker used as a reference for fragment size
C) A staining reagent
D) The wells in the gel

*Answer: B) A DNA marker used as a reference for fragment size

*Question: What do smeared or smudged bands in a gel electrophoresis result indicate?
A) Successful amplification
B) High DNA concentration
C) Degraded DNA, destroyed DNA, or contaminants present
D) Low voltage used

*Answer: C) Degraded DNA, destroyed DNA, or contaminants present

*Question: What does a single sharp band in gel electrophoresis at the expected amplicon size indicate?
A) Contamination
B) Non-specific amplification
C) Successful and specific amplification
D) Failed PCR

*Answer: C) Successful and specific amplification

*Question: Why would an unexpected band appear at a different size than expected in gel electrophoresis?
A) Too much magnesium
B) The primer was not specific to the target gene
C) Too little buffer
D) Incorrect voltage

*Answer: B) The primer was not specific to the target gene

*Question: Which sample shows the MOST successful and specific amplification in gel electrophoresis?
A) A sample with multiple bands
B) A sample with smeared bands
C) A sample with only ONE band at the expected amplicon size
D) A sample with no bands

*Answer: C) A sample with only ONE band at the expected amplicon size

*Question: What is the purpose of a negative control in PCR/gel electrophoresis?
A) To confirm primer specificity and detect contamination
B) To serve as the DNA template
C) To increase the number of amplicons
D) To calibrate the thermal cycler

*Answer: A) To confirm primer specificity and detect contamination

*Question: What components are included in the negative control?
A) All PCR components including DNA template
B) DNA template only, no polymerase
C) All PCR components EXCEPT the DNA template
D) Only buffer and dNTPs

*Answer: C) All PCR components EXCEPT the DNA template

*Question: What is the expected result of a negative control in gel electrophoresis?
A) A bright band at the expected amplicon size
B) Multiple bands indicating amplification
C) No band (negative result)
D) A smeared result

*Answer: C) No band (negative result)

*Question: What does it mean if a band appears in the negative control lane during gel electrophoresis?
A) The PCR was successful
B) The DNA concentration is high
C) The PCR is INVALIDATED due to contamination; the run must be repeated
D) The ladder was loaded incorrectly

*Answer: C) The PCR is INVALIDATED due to contamination; the run must be repeated

*Question: Why is a positive control considered optional in gel electrophoresis (per the lecture)?
A) It serves no purpose
B) It is expensive and gel electrophoresis with the ladder is already size-specific
C) It gives false results
D) It is replaced by the negative control

*Answer: B) It is expensive and gel electrophoresis with the ladder is already size-specific

*Question: The negative control and the actual sample must be processed:
A) On different days
B) At the same time
C) With different primers
D) At different temperatures

*Answer: B) At the same time

*Question: In real-time PCR, what happens to samples whose amplification occurs BEYOND the set number of cycles?
A) They are considered positive results
B) They can be considered negative control, contaminated, or nonspecific amplification — they are rejected
C) They are retested with a lower CT value
D) They represent the highest DNA concentration

*Answer: B) They can be considered negative control, contaminated, or nonspecific amplification — they are rejected