HYBRIDIZATION

*Question: What is the fundamental principle of nucleic acid hybridization?
A) Double-stranded nucleic acids bind to complementary sequences
B) Single-stranded nucleic acids bind to complementary sequences
C) RNA polymerase binds to DNA promoter regions
D) Restriction enzymes cut DNA at specific sites

*Answer: B) Single-stranded nucleic acids bind to complementary sequences

*Question: Which of the following correctly lists the types of hybrids formed in nucleic acid hybridization?
A) DNA–DNA, DNA–Protein, RNA–RNA
B) DNA–DNA, DNA–RNA, RNA–RNA
C) RNA–RNA, RNA–Protein, DNA–DNA
D) DNA–DNA, RNA–Protein, DNA–RNA

*Answer: B) DNA–DNA, DNA–RNA, RNA–RNA

*Question: In the hybrid format notation (e.g., DNA–RNA), what does the first molecule represent?
A) The probe
B) The label
C) The original/source/target
D) The membrane

*Answer: C) The original/source/target

*Question: What is a PROBE in nucleic acid hybridization?
A) A double-stranded DNA fragment used to amplify sequences
B) A single-stranded nucleic acid bearing the complementary base sequence of the target
C) An enzyme that cuts DNA at palindromic sites
D) A fluorescent membrane used to detect DNA

*Answer: B) A single-stranded nucleic acid bearing the complementary base sequence of the target

*Question: How does nucleic acid hybridization (NAH) differ from PCR in its second step?
A) PCR uses renaturation; NAH uses annealing
B) PCR uses a probe; NAH uses a primer
C) PCR uses annealing with a primer; NAH uses renaturation with a probe
D) Both use identical steps in the second phase

*Answer: C) PCR uses annealing with a primer; NAH uses renaturation with a probe

*Question: What step do both PCR and nucleic acid hybridization share?
A) Annealing
B) Denaturation
C) Renaturation
D) Ligation

*Answer: B) Denaturation

*Question: What is the standard optimum denaturation temperature in both PCR and NAH?
A) 65°C
B) 72°C
C) 95°C
D) 37°C

*Answer: C) 95°C

*Question: Why does a nucleic acid rich in G and C content require a higher denaturation temperature?
A) G-C pairs form only 1 hydrogen bond
B) G-C pairs form 2 hydrogen bonds
C) G-C pairs form 3 hydrogen bonds
D) G-C pairs are held together by covalent bonds

*Answer: C) G-C pairs form 3 hydrogen bonds

*Question: Which chemical can be used as an alternative to heat for denaturation in NAH?
A) Ethanol
B) Formamide
C) Sodium chloride
D) Tetrazolium dye

*Answer: B) Formamide

*Question: What temperature is typically used to facilitate renaturation/reannealing in NAH?
A) 95°C
B) 72°C
C) 37°C
D) 65°C

*Answer: D) 65°C

*Question: What is the correct order of steps in nucleic acid hybridization?
A) Hybridization → Denaturation → Renaturation
B) Renaturation → Denaturation → Hybridization
C) Denaturation → Renaturation → Hybridization
D) Denaturation → Hybridization → Renaturation

*Answer: C) Denaturation → Renaturation → Hybridization

*Question: What term describes raw, unamplified DNA directly from a sample?
A) Amplicons
B) Genomic DNA (gDNA)
C) cDNA
D) Plasmid DNA

*Answer: B) Genomic DNA (gDNA)

*Question: What term is used for DNA that has been amplified by PCR?
A) Genomic DNA
B) Oligonucleotides
C) PCR products or amplicons
D) Recombinant DNA

*Answer: C) PCR products or amplicons

*Question: Which detection methods can be used to visualize the target sequence after hybridization?
A) Mass spectrometry and HPLC
B) X-ray, autoradiography, and fluorescence microscopy
C) Gel electrophoresis only
D) Western blotting only

*Answer: B) X-ray, autoradiography, and fluorescence microscopy

*Question: What is the role of the TARGET in nucleic acid hybridization?
A) It carries the label and complementary bases
B) It is a single-stranded probe free in solution
C) It is the denatured DNA or RNA containing the gene of interest
D) It is the restriction enzyme that cuts the DNA

*Answer: C) It is the denatured DNA or RNA containing the gene of interest

*Question: Why does RNA not need denaturation before hybridization when used as a target?
A) RNA is already double-stranded
B) RNA is already single-stranded
C) RNA is heat-resistant
D) RNA does not bind to probes

*Answer: B) RNA is already single-stranded

*Question: To which solid surfaces can a target be bound during hybridization?
A) Glass slide and agarose gel
B) Nylon or nitrocellulose membrane
C) Polyacrylamide gel
D) Polystyrene plate

*Answer: B) Nylon or nitrocellulose membrane

*Question: What is attached to the probe to make it detectable?
A) A palindromic sequence
B) A signal-producing moiety (label)
C) A restriction enzyme
D) A polymerase

*Answer: B) A signal-producing moiety (label)

*Question: What does a longer probe sequence offer compared to a shorter one?
A) Higher background noise and less specificity
B) Better detection of single base mutations
C) Greater specificity and better complementarity with the target
D) Faster hybridization speed

*Answer: C) Greater specificity and better complementarity with the target

*Question: When are shorter probes preferred in NAH?
A) When detecting long genomic sequences
B) When performing mutational analyses, especially point mutations
C) When high GC content is present
D) When using radioisotopes as labels

*Answer: B) When performing mutational analyses, especially point mutations

*Question: What is the main drawback of using shorter probes?
A) They cannot bind to RNA
B) They have high background noise due to nonspecific binding
C) They require higher denaturation temperatures
D) They cannot be labeled with fluorophores

*Answer: B) They have high background noise due to nonspecific binding

*Question: Which early method of probe production involves using E. coli plasmid as a vector?
A) In vitro organic synthesis
B) PCR amplification
C) Cloning into a plasmid
D) Viral genome isolation

*Answer: C) Cloning into a plasmid

*Question: What is the purpose of restriction enzyme digestion in early probe production?
A) To label the probe with radioisotopes
B) To cleave long strands of DNA into shorter fragments usable as probes
C) To perform denaturation of the double-stranded DNA
D) To transfer DNA onto a nitrocellulose membrane

*Answer: B) To cleave long strands of DNA into shorter fragments usable as probes

*Question: Which restriction enzymes are examples of those used in probe production and Southern blotting?
A) DNA polymerase I and T4 ligase
B) EcoRI, BamHI, HindIII
C) Terminal transferase and T4 polynucleotide kinase
D) Alkaline phosphatase and horseradish peroxidase

*Answer: B) EcoRI, BamHI, HindIII

*Question: What is a palindromic sequence in double-stranded DNA?
A) A sequence that codes for restriction enzymes
B) A sequence that reads the same in the 5'→3' direction on both strands due to antiparallelism
C) A repetitive sequence found only in eukaryotic DNA
D) A sequence that prevents hybridization

*Answer: B) A sequence that reads the same in the 5'→3' direction on both strands due to antiparallelism

*Question: Which of the following is a NEW method of DNA probe production?
A) Gel purification from agarose gels
B) Cloning into a plasmid
C) In vitro organic synthesis of short oligomeric probes
D) Isolation by restriction enzyme digestion

*Answer: C) In vitro organic synthesis of short oligomeric probes

*Question: How are RNA probes produced?
A) Through direct PCR amplification of RNA
B) Through in vitro transcription from a DNA template (plasmid or PCR amplicons)
C) Through restriction enzyme digestion of RNA
D) Through Southern blotting

*Answer: B) Through in vitro transcription from a DNA template (plasmid or PCR amplicons)

*Question: How does the sensitivity of RNA probes compare to DNA probes?
A) RNA probes are less sensitive than DNA probes
B) RNA probes have equal or greater sensitivity than DNA probes
C) RNA and DNA probes have identical sensitivity
D) DNA probes are always more sensitive

*Answer: B) RNA probes have equal or greater sensitivity than DNA probes

*Question: Why are RNA probes considered more sensitive?
A) They are double-stranded and more stable
B) They are single-stranded and do not need denaturation, allowing easy binding to the target
C) They emit higher fluorescence signals
D) They have radioactive labels

*Answer: B) They are single-stranded and do not need denaturation, allowing easy binding to the target

*Question: What is the promoter region sequence in eukaryotes associated with transcription?
A) PRIBNOW box (TATAAT)
B) TATA box (TATA)
C) Shine-Dalgarno sequence
D) Kozak sequence

*Answer: B) TATA box (TATA)

*Question: Which RNA probe production step involves inserting a different gene into the vector?
A) Linearization of the vector
B) Transcription from the promoter
C) Formation of a recombinant vector
D) Labeling of the probe

*Answer: C) Formation of a recombinant vector

*Question: Which labeling method adds labeled molecules to the 5' or 3' end of the probe using enzymes?
A) Nick translation
B) Random priming
C) End labeling
D) In vitro transcription

*Answer: C) End labeling

*Question: Which enzymes are used in end labeling of probes?
A) DNA Polymerase I and RNA Polymerase
B) Terminal transferase or T4 polynucleotide kinase
C) Alkaline phosphatase and HRP
D) EcoRI and BamHI

*Answer: B) Terminal transferase or T4 polynucleotide kinase

*Question: In nick translation, what are "nicks"?
A) Cuts made by restriction enzymes at palindromic sequences
B) Shallow single-stranded breaks or strand breaks in the DNA probe
C) Gaps in the RNA probe caused by RNase activity
D) Double-stranded breaks caused by UV radiation

*Answer: B) Shallow single-stranded breaks or strand breaks in the DNA probe

*Question: In nick translation, which enzyme adds labeled nucleotides to extend the nick?
A) RNA polymerase
B) Terminal transferase
C) DNA polymerase
D) T4 ligase

*Answer: C) DNA polymerase

*Question: What is random priming in probe labeling?
A) Randomly attaching radioisotopes to the 3' end of DNA
B) A short probe hybridizes with ssDNA and labeled dNTPs are added during extension, generating a new labeled probe
C) Adding labels to nicks in double-stranded DNA using DNA polymerase
D) UV crosslinking of the probe to the nitrocellulose membrane

*Answer: B) A short probe hybridizes with ssDNA and labeled dNTPs are added during extension, generating a new labeled probe

*Question: Which radioisotopes are commonly used to label probes?
A) Carbon-14 and Tritium
B) Phosphorus-32, Sulfur-35, and Nitrogen
C) Iodine-131 and Uranium-238
D) Potassium-40 and Calcium-45

*Answer: B) Phosphorus-32, Sulfur-35, and Nitrogen

*Question: Why are radioisotopes no longer favored as probe labels?
A) They are too expensive and unavailable
B) They are hazardous to humans and the environment, and have short half-lives
C) They cannot be detected by autoradiography
D) They produce too much background noise

*Answer: B) They are hazardous to humans and the environment, and have short half-lives

*Question: How are radio-labeled probes detected after hybridization?
A) Fluorescence microscope
B) Mass spectrometry
C) Autoradiography or X-ray film
D) Chemiluminescence only

*Answer: C) Autoradiography or X-ray film

*Question: What are the most common non-radioactive labels used in probe labeling?
A) Fluorescein and rhodamine
B) Biotin and digoxygenin attached to UTP or CTP
C) Phosphorus-32 and Sulfur-35
D) Ethidium bromide and SYBR Green

*Answer: B) Biotin and digoxygenin attached to UTP or CTP

*Question: Which molecules are conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP) for detection of non-radioactive labels?
A) Streptavidin (biotin) or antidigoxygenin (digoxygenin)
B) T4 polynucleotide kinase and terminal transferase
C) EcoRI and BamHI
D) Fluorophore and quencher

*Answer: A) Streptavidin (biotin) or antidigoxygenin (digoxygenin)

*Question: What substrates are used for chemiluminescent detection of non-radioactive labels?
A) Ethidium bromide and agarose
B) Dioxetane or tetrazolium dye
C) Formamide and NaOH
D) SSC buffer and EDTA

*Answer: B) Dioxetane or tetrazolium dye

*Question: What is the role of fluorophores in probe labeling?
A) They cut DNA at specific palindromic sequences
B) They absorb light at a specific wavelength and emit vivid fluorescent light for detection
C) They suppress nonspecific binding of the probe
D) They transfer DNA from the gel to the membrane

*Answer: B) They absorb light at a specific wavelength and emit vivid fluorescent light for detection

*Question: What is the function of the quencher molecule in fluorescent probe labeling?
A) It amplifies the fluorescence signal from the probe
B) It suppresses emission of light from background noise and nonspecific sequences
C) It labels the 5' end of the probe
D) It conjugates the probe to the nylon membrane

*Answer: B) It suppresses emission of light from background noise and nonspecific sequences

*Question: How are fluorescently labeled probes detected?
A) Autoradiography
B) X-ray film
C) Fluorescence microscope
D) Chemiluminescence

*Answer: C) Fluorescence microscope

*Question: Which of the following is NOT a non-radioactive label used in NAH?
A) Biotin
B) Digoxygenin
C) Fluorescent dye
D) Phosphorus-32

*Answer: D) Phosphorus-32

*Question: To which nucleotides are biotin and digoxygenin typically attached in non-radioactive labeling?
A) ATP or GTP
B) UTP or CTP
C) dATP or dGTP
D) tRNA or rRNA

*Answer: B) UTP or CTP

*Question: What are the four main categories of hybridization techniques?
A) Solid-phase, Solution-phase, In situ, and Gel-based
B) Radioisotope, Fluorescent, Enzymatic, and Chemical
C) Southern, Northern, Western, and Eastern
D) Capillary, Vacuum, Electrophoretic, and Gel

*Answer: A) Solid-phase, Solution-phase, In situ, and Gel-based

*Question: Which hybridization technique category includes Southern and Northern blotting?
A) Solution-phase hybridization
B) In situ hybridization
C) Solid-phase hybridization
D) Gel-based hybridization

*Answer: C) Solid-phase hybridization

*Question: Which technique is classified under solution-phase hybridization?
A) FISH
B) SSCP
C) FRET (Fluorescence Resonance Energy Transfer)
D) Southern blot

*Answer: C) FRET (Fluorescence Resonance Energy Transfer)

*Question: What is the target molecule in a Southern blot?
A) RNA
B) Protein
C) DNA
D) Lipids

*Answer: C) DNA

*Question: What is the target molecule in a Northern blot?
A) DNA
B) Protein
C) Lipids
D) RNA

*Answer: D) RNA

*Question: What is the target molecule in a Western blot?
A) DNA
B) RNA
C) Protein
D) Lipids

*Answer: C) Protein

*Question: What is the target in a Southwestern blot?
A) RNA
B) Lipids
C) DNA
D) Protein

*Answer: D) Protein

*Question: What is the PURPOSE of a Southwestern blot?
A) Studying RNA structure and gene expression
B) Detecting DNA binding proteins and gene expression
C) Detecting lipids via mass spectrometry
D) Studying protein processing

*Answer: B) Detecting DNA binding proteins and gene expression

*Question: Who first developed the Southern blot technique?
A) James Watson
B) Frederick Sanger
C) Edwin Southern
D) Kary Mullis

*Answer: C) Edwin Southern

*Question: What is the primary use of Southern blotting?
A) Studying RNA structure and transcript processing
B) Studying gene structure and detection of DNA sequences in a sample
C) Detecting protein modifications
D) Analyzing lipid composition

*Answer: B) Studying gene structure and detection of DNA sequences in a sample

*Question: What is the correct order of steps in Southern blotting?
A) Blotting → RE digestion → Resolution → Denaturation → Probing → Detection
B) RE digestion → Resolution → Denaturation → Blotting → Probing → Detection
C) Denaturation → Blotting → RE digestion → Resolution → Probing → Detection
D) Resolution → RE digestion → Denaturation → Probing → Blotting → Detection

*Answer: B) RE digestion → Resolution → Denaturation → Blotting → Probing → Detection

*Question: What recognition sites do restriction endonucleases typically have?
A) 1–2 bp random sequences
B) 4–6 bp palindromic sequences
C) 10–12 bp repetitive sequences
D) 20–30 bp promoter sequences

*Answer: B) 4–6 bp palindromic sequences

*Question: What is "star activity" in restriction enzyme digestion?
A) Enhanced cutting efficiency under optimal conditions
B) Altered enzyme specificity under nonstandard reaction conditions
C) Radioactive labeling of restriction enzymes
D) Nonspecific fluorescence of gel-resolved fragments

*Answer: B) Altered enzyme specificity under nonstandard reaction conditions

*Question: What is a Restriction Fragment Length Polymorphism (RFLP)?
A) A type of probe used in Southern blotting
B) Variation in the number and location of restriction sites among individuals
C) A palindromic recognition sequence shared by all restriction enzymes
D) A technique used to label probes with radioisotopes

*Answer: B) Variation in the number and location of restriction sites among individuals

*Question: How are DNA fragments resolved after restriction enzyme digestion in Southern blotting?
A) Capillary transfer
B) Gel electrophoresis
C) Vacuum blotting
D) Ultracentrifugation

*Answer: B) Gel electrophoresis

*Question: In gel electrophoresis for Southern blotting, how do larger DNA fragments migrate?
A) Faster toward the anode
B) Faster toward the cathode
C) Slower toward the anode
D) They do not migrate

*Answer: C) Slower toward the anode

*Question: What does a smear in the lower region of a Southern blot gel indicate?
A) Incomplete digestion
B) Star activity
C) Degraded DNA
D) Successful hybridization

*Answer: C) Degraded DNA

*Question: What do large aggregates near the top of a Southern blot gel indicate?
A) Degraded DNA
B) Incomplete digestion
C) Successful resolution
D) Nonspecific probe binding

*Answer: B) Incomplete digestion

*Question: In Southern blotting, what chemical is used directly for denaturation of DNA fragments?
A) Formamide
B) Ethanol
C) NaOH (sodium hydroxide)
D) Acetone

*Answer: C) NaOH (sodium hydroxide)

*Question: When is HCl used before denaturation in Southern blotting?
A) For all DNA fragments regardless of size
B) For depurination prior to denaturation for fragments >500 bp
C) Only for RNA targets
D) When using vacuum transfer

*Answer: B) For depurination prior to denaturation for fragments >500 bp

*Question: What is the purpose of the blotting/transfer step in Southern blotting?
A) To amplify the DNA fragments before probing
B) To move denatured DNA from the gel to a solid membrane for probing
C) To label the DNA fragments with radioisotopes
D) To perform restriction enzyme digestion

*Answer: B) To move denatured DNA from the gel to a solid membrane for probing

*Question: In capillary transfer, the gel is placed on top of which solution?
A) 10X phosphate buffer
B) 10X saline sodium citrate (SSC)
C) 20X EDTA buffer
D) Formaldehyde solution

*Answer: B) 10X saline sodium citrate (SSC)

*Question: How long does capillary transfer typically take?
A) 30 minutes
B) 2–3 hours
C) Approximately 16 hours (overnight)
D) 5 minutes

*Answer: C) Approximately 16 hours (overnight)

*Question: According to the mnemonic, in capillary transfer, what goes on TOP?
A) The membrane
B) The buffer
C) The gel
D) The absorbent paper

*Answer: C) The gel

*Question: What is the mnemonic for the capillary transfer process?
A) BL BWH
B) AML (Absorb, Move, Left overnight)
C) De-Good-Doesn't Blur-A Clear Base Sequence
D) GEL ON TOP

*Answer: B) AML (Absorb, Move, Left overnight)

*Question: Which transfer method uses electric current to carry DNA and is the FASTEST?
A) Capillary transfer
B) Vacuum transfer
C) Electrophoretic transfer
D) Gravity transfer

*Answer: C) Electrophoretic transfer

*Question: In vacuum transfer, what is placed FIRST before the gel?
A) The buffer
B) The membrane
C) The absorbent paper
D) The gel

*Answer: B) The membrane

*Question: How long does vacuum transfer typically take?
A) Overnight (~16 hours)
B) 5–10 minutes
C) 2–3 hours
D) 48 hours

*Answer: C) 2–3 hours

*Question: What is the purpose of the PREHYBRIDIZATION step in Southern blotting?
A) To denature the DNA fragments before blotting
B) To prevent the probe from binding nonspecifically to the membrane
C) To label the probe with a fluorescent dye
D) To wash off unbound DNA fragments

*Answer: B) To prevent the probe from binding nonspecifically to the membrane

*Question: How is DNA immobilized to the membrane in vacuum transfer?
A) Capillary action and gravity
B) Baking in a vacuum oven or UV crosslinking
C) Electric current application
D) Salt precipitation

*Answer: B) Baking in a vacuum oven or UV crosslinking

*Question: What is the purpose of the washing step after probe hybridization?
A) To denature the remaining double-stranded DNA
B) To remove unbound or excess labeled probes to prevent nonspecific binding
C) To transfer DNA from the gel to the membrane
D) To apply the substrate for chemiluminescent detection

*Answer: B) To remove unbound or excess labeled probes to prevent nonspecific binding

*Question: What does FISH stand for?
A) Fluorescent Imaging of Sequence Hybridization
B) Fluorescence In Situ Hybridization
C) Fragment Identification by Sequence Homology
D) Functional In Situ Hybridization

*Answer: B) Fluorescence In Situ Hybridization

*Question: What type of probe label does FISH use?
A) Radioisotope (32P)
B) Biotin attached to CTP
C) Fluorescent-labeled probes
D) Digoxygenin attached to UTP

*Answer: C) Fluorescent-labeled probes

*Question: What can FISH detect that makes it clinically significant?
A) Specific mRNA expression in gels
B) Chromosomal abnormalities such as translocations (e.g., Philadelphia chromosome in leukemia)
C) Protein modifications in Western blots
D) Lipid composition in cell membranes

*Answer: B) Chromosomal abnormalities such as translocations (e.g., Philadelphia chromosome in leukemia)

*Question: In FISH, what does fluorescence at a specific chromosomal region indicate?
A) Normal gene expression
B) Successful PCR amplification
C) An abnormality or mutation at that chromosomal location
D) Successful restriction enzyme digestion

*Answer: C) An abnormality or mutation at that chromosomal location

*Question: What is the target in Northern blotting?
A) DNA
B) Protein
C) RNA (mRNA)
D) Lipids

*Answer: C) RNA (mRNA)

*Question: What is Northern blotting primarily used to study?
A) Gene structure and restriction fragment patterns
B) RNA structure, gene expression, and mutational analyses
C) Protein processing and post-translational modifications
D) Lipid transfer and mass spectrometry analysis

*Answer: B) RNA structure, gene expression, and mutational analyses

*Question: Which key difference does Northern blotting have from Southern blotting regarding denaturation?
A) Northern blotting uses heat for denaturation
B) Northern blotting has no separate denaturation step — AGE is run under denaturing conditions
C) Northern blotting uses NaOH for a separate denaturation step
D) Northern blotting skips the gel electrophoresis step entirely

*Answer: B) Northern blotting has no separate denaturation step — AGE is run under denaturing conditions

*Question: What denaturing agents are used in the gel conditions for Northern blotting?
A) NaOH and HCl
B) Formaldehyde or glyoxal
C) Ethidium bromide and SYBR Green
D) Sodium chloride and EDTA

*Answer: B) Formaldehyde or glyoxal

*Question: What special environment must be maintained during Northern blotting?
A) Sterile environment with no bacteria
B) RNase-free environment
C) DNase-free environment
D) High salt concentration environment

*Answer: B) RNase-free environment

*Question: What SSC concentration is used in Northern blotting compared to Southern blotting?
A) Northern uses 10X SSC; Southern uses 20X SSC
B) Both use the same 10X SSC concentration
C) Northern uses 20X SSC; Southern uses 10X SSC
D) Northern uses 5X SSC; Southern uses 10X SSC

*Answer: C) Northern uses 20X SSC; Southern uses 10X SSC

*Question: Why is Northern blotting considered more sensitive than Southern blotting?
A) It uses more concentrated probes
B) RNA is already single-stranded, requiring no denaturation and allowing easier probe binding
C) Northern blotting uses radioisotopes exclusively
D) It uses higher voltage during electrophoresis

*Answer: B) RNA is already single-stranded, requiring no denaturation and allowing easier probe binding

*Question: What is STRINGENCY in the context of nucleic acid hybridization?
A) The length of the probe used in hybridization
B) The degree of homology between the probe and the nucleic acid target
C) The concentration of salt in the washing buffer
D) The temperature used for restriction enzyme digestion

*Answer: B) The degree of homology between the probe and the nucleic acid target

*Question: What happens when stringency is TOO HIGH during hybridization?
A) Nonspecific binding occurs
B) No binding will occur at all
C) Background noise increases
D) The probe degrades

*Answer: B) No binding will occur at all

*Question: What happens when stringency is TOO LOW during hybridization?
A) No binding occurs
B) The probe is degraded by RNases
C) Nonspecific binding occurs
D) DNA fragments migrate too slowly

*Answer: C) Nonspecific binding occurs

*Question: Which TWO factors affect stringency in hybridization?
A) Probe length and membrane type
B) Temperature and salt concentration (ionic strength)
C) Gel voltage and buffer pH
D) Probe type and restriction enzyme used

*Answer: B) Temperature and salt concentration (ionic strength)

*Question: For a perfect match sequence requiring high stringency, what conditions are needed?
A) Low temperature and high salt concentration
B) High temperature and high salt concentration
C) High temperature and low salt concentration
D) Low temperature and low salt concentration

*Answer: C) High temperature and low salt concentration

*Question: Why does low salt concentration increase stringency?
A) Salt stabilizes the DNA by shielding negative charges; low salt compromises DNA stability making denaturation easier
B) Salt blocks probe binding; low salt allows more probe binding
C) Salt increases fluorescence; low salt reduces background noise
D) Salt activates restriction enzymes; low salt deactivates them

*Answer: A) Salt stabilizes the DNA by shielding negative charges; low salt compromises DNA stability making denaturation easier

*Question: What confers the negative charge on DNA?
A) Deoxyribose sugar
B) Nitrogenous bases
C) Phosphate groups
D) Hydrogen bonds

*Answer: C) Phosphate groups

*Question: What is the formula for calculating the melting temperature (Tm) of DNA?
A) Tm = 72°C + 0.41(%GC) − 600/n
B) Tm = 81.5°C + 16.6 log M + 0.41(%GC) − 0.61(% formamide) − 600/n
C) Tm = 95°C − 0.61(%GC) + 16.6 log M
D) Tm = 65°C + 0.41(%GC) − 16.6 log M

*Answer: B) Tm = 81.5°C + 16.6 log M + 0.41(%GC) − 0.61(% formamide) − 600/n

*Question: What is the optimal hybridization temperature relative to Tm?
A) Exactly at Tm
B) 5°C above Tm
C) 5°C below Tm
D) 25°C above Tm

*Answer: C) 5°C below Tm

*Question: How much does RNA:DNA hybrid formation increase Tm compared to DNA:DNA?
A) 5–10°C
B) 10–15°C
C) 20–25°C
D) 30–35°C

*Answer: B) 10–15°C

*Question: How much does RNA:RNA hybrid formation increase Tm?
A) 5–10°C
B) 10–15°C
C) 20–25°C
D) 30–35°C

*Answer: C) 20–25°C