Mol Diag Ch 5 (MC convert)

What type of restriction enzyme is commonly used in the laboratory?
(A) Type I, 20+ bp recognition sites
(B) Type II, 4-6 bp recognition sites
(C) Type III, 10-12 bp recognition sites
(D) Type IV, methylation-sensitive enzymes

B. Type II, 4-6 bp recognition sites

What is a restriction map?
(A) A diagram showing DNA replication sites
(B) A diagram showing the lengths of fragments between restriction sites in DNA
(C) A map showing the location of genes on chromosomes
(D) A map showing RNA polymerase binding sites

B. A diagram showing the lengths of fragments between restriction sites in DNA

If you cut a linear fragment of DNA with a restriction enzyme at three sites, how many fragments do you get? What about if you cut a plasmid at three sites?
(A) 3 fragments linear, 4 fragments plasmid
(B) 4 fragments linear, 3 fragments plasmid
(C) 3 fragments linear, 3 fragments plasmid
(D) 4 fragments linear, 4 fragments plasmid

B. 4 fragments linear, 3 fragments plasmid

How do you perform restriction mapping?
(A) Sequence the DNA directly
(B) Cut DNA with one restriction enzyme only
(C) Cut DNA with increasing combinations of restriction enzymes and use fragment sizes to locate cut sites
(D) Use PCR to amplify cut sites

C. Cut DNA with increasing combinations of restriction enzymes and use fragment sizes to locate cut sites

Are restriction enzyme cuts unique to different strands of nucleic acid?
(A) No, they cut identical sequences in all individuals
(B) Yes, due to somatic differences; fragment analysis can be used for paternity or forensic profiling
(C) No, they cut random sequences
(D) Yes, but only in prokaryotes

B. Yes, due to somatic differences; fragment analysis can be used for paternity or forensic profiling

What is star activity in restriction enzymes?
(A) Increased enzyme specificity under ideal conditions
(B) When restriction enzymes cut at incorrect sequences due to prolonged reaction or wrong buffer
(C) Enzymes losing all activity after freezing
(D) Enzyme cutting only methylated DNA

B. When restriction enzymes cut at incorrect sequences due to prolonged reaction or wrong buffer

What are Restriction Fragment Length Polymorphisms (RFLPs)?
(A) Differences in DNA sequence causing different patterns of fragment lengths after enzyme digestion
(B) RNA fragments produced during transcription
(C) Mutations in the promoter region only
(D) DNA sequences identical across homologous chromosomes

A. Differences in DNA sequence causing different patterns of fragment lengths after enzyme digestion

What does CRISPR stand for?
(A) Clustered regularly interspaced short palindromic repeats
(B) Chromosomal repeated interspersed sequences
(C) Cytoplasmic RNA sequences
(D) Cell regulated integration system

A. Clustered regularly interspaced short palindromic repeats

What is the main difference between CRISPR/Cas9 system DNA cuts and restriction enzymes?
(A) CRISPR cuts random DNA, restriction enzymes cut RNA
(B) Restriction enzymes cut specific DNA sequences; CRISPR/Cas9 cuts RNA only
(C) Restriction enzymes cut at enzyme recognition sites; CRISPR/Cas9 cuts DNA guided by RNA which can be designed
(D) Both cut at identical sites but use different proteins

C. Restriction enzymes cut at enzyme recognition sites; CRISPR/Cas9 cuts DNA guided by RNA which can be designed

What is Cas9?
(A) DNA polymerase involved in replication
(B) RNA-guided DNA endonuclease enzyme associated with CRISPR that produces single strand breaks
(C) A type of restriction enzyme
(D) A transcription factor

B. RNA-guided DNA endonuclease enzyme associated with CRISPR that produces single strand breaks

What is crRNA in the CRISPR system?
(A) CRISPR RNA derived from spacer element that matches target DNA to cut
(B) Protein that cleaves DNA
(C) DNA sequence that binds Cas9
(D) RNA polymerase involved in transcription

A. CRISPR RNA derived from spacer element that matches target DNA to cut

What is tracrRNA?
(A) Transfer RNA that helps in protein synthesis
(B) Trans Activating CRISPR RNA, required for binding to Cas9 protein
(C) RNA that degrades viral DNA
(D) RNA that serves as a primer for DNA replication

B. Trans Activating CRISPR RNA, required for binding to Cas9 protein

What is the PAM sequence?
(A) Protospacer Adjacent Motif, necessary sequence adjacent to target DNA for CRISPR to discriminate target from self
(B) A site where DNA polymerase binds
(C) RNA sequence complementary to crRNA
(D) Restriction enzyme recognition site

A. Protospacer Adjacent Motif, necessary sequence adjacent to target DNA for CRISPR to discriminate target from self

What is a Southern Blot?
(A) A method to measure RNA expression levels
(B) A DNA sample cut with restriction enzymes, separated by gel electrophoresis, transferred to a membrane, and probed with labeled DNA to detect specific sequences
(C) A protein detection method using antibodies
(D) A technique for DNA sequencing

B. A DNA sample cut with restriction enzymes, separated by gel electrophoresis, transferred to a membrane, and probed with labeled DNA to detect specific sequences

What is Nitrocellulose used for in blotting?
(A) To stain DNA in gels
(B) To act as a paper membrane that promotes probe binding by attracting DNA/RNA through positive charge
(C) To digest proteins during blotting
(D) To amplify DNA fragments

B. To act as a paper membrane that promotes probe binding by attracting DNA/RNA through positive charge

Is Southern blot analysis used for small or large regions of DNA?
(A) Small regions (~100 bp)
(B) Large regions (10-100 kb)
(C) Only mitochondrial DNA
(D) Only RNA molecules

B. Large regions (10-100 kb)

For large fragments in gel electrophoresis, compared to smaller fragments, the run time is and the voltage is .
(A) shorter, higher
(B) shorter, lower
(C) longer, lower
(D) longer, higher

C. longer, lower

After restriction enzyme digestion of DNA, how should bands look on a gel?
(A) A single sharp band
(B) Even smear indicates successful digestion; a large band at the start indicates incomplete digestion; a large band at the end indicates degraded DNA
(C) Multiple bands of the same size
(D) No bands visible

B. Even smear indicates successful digestion; a large band at the start indicates incomplete digestion; a large band at the end indicates degraded DNA

How do you distinguish the target DNA fragment from other fragments in Southern blot analysis?
(A) By color staining of all DNA
(B) Through hybridization of a complementary DNA or RNA probe
(C) By size alone
(D) Using antibodies

B. Through hybridization of a complementary DNA or RNA probe

How are large DNA fragments prepared prior to denaturation in Southern blot analysis?
(A) By depurination (removal of purine bases using HCl) to loosen DNA
(B) By heating at 100°C
(C) By digestion with proteases
(D) By adding salt

A. By depurination (removal of purine bases using HCl) to loosen DNA

How is DNA denatured prior to blotting in Southern blot analysis?
(A) Soaked in strong acid (HCl)
(B) Soaked in strong base (NaOH) which breaks hydrogen bonds
(C) Heated to 37°C
(D) Treated with protease

B. Soaked in strong base (NaOH) which breaks hydrogen bonds

Is nucleic acid permanently bound to nitrocellulose after transfer? What is the advantage?
(A) No, it can easily wash away
(B) Yes; allows multiple probes to be used since probes can be removed and reapplied
(C) Yes; but only for RNA
(D) No; it only temporarily sticks

B. Yes; allows multiple probes to be used since probes can be removed and reapplied

What happens if the nitrocellulose membrane isn’t soaked evenly in buffer?
(A) Probe binding won’t occur in dry areas, causing uneven signals
(B) The entire membrane will be damaged
(C) DNA will degrade
(D) Binding will be uniform anyway

A. Probe binding won’t occur in dry areas, causing uneven signals

What is capillary transfer in blotting?
(A) Transfer using an electric current
(B) Transfer of nucleic acid from gel to membrane using capillary action through buffer-soaked papers
(C) Transfer using vacuum suction
(D) Transfer by diffusion in liquid solution

B. Transfer of nucleic acid from gel to membrane using capillary action through buffer-soaked papers

What is electrophoretic transfer in blotting?
(A) Using suction to transfer DNA
(B) Using electric current to move DNA from gel (cathode) to membrane (anode) in buffer tank
(C) Using capillary action with paper
(D) Using magnetic beads

B. Using electric current to move DNA from gel (cathode) to membrane (anode) in buffer tank

What is vacuum transfer?
(A) Transfer of DNA using suction and recirculating buffer, faster but requires specialized equipment
(B) Transfer by heating the membrane
(C) Transfer by shaking the gel
(D) Transfer using magnetic fields

A. Transfer of DNA using suction and recirculating buffer, faster but requires specialized equipment

How is DNA permanently immobilized on the membrane after transfer in Southern blot?
(A) Heating (baking) or UV cross-linking
(B) Washing with ethanol
(C) Staining with dyes
(D) Adding proteases

A. Heating (baking) or UV cross-linking

What is the purpose of prehybridization in Southern blot analysis?
(A) To degrade non-target DNA
(B) To prevent nonspecific binding of probe
(C) To stain DNA
(D) To amplify DNA fragments

B. To prevent nonspecific binding of probe

Which of the following are examples of prehybridization buffers?
(A) Ficoll, polyvinyl pyrrolidine, bovine serum albumin, salmon sperm DNA, SDS, formamide
(B) Ethanol, methanol, chloroform
(C) Water only
(D) Acetic acid

A. Ficoll, polyvinyl pyrrolidine, bovine serum albumin, salmon sperm DNA, SDS, formamide

What is the Northern Blot technique used for?
(A) DNA analysis with restriction enzymes
(B) RNA analysis using DNA probes; analyzes gene expression and mRNA abnormalities
(C) Protein detection
(D) DNA sequencing

B. RNA analysis using DNA probes; analyzes gene expression and mRNA abnormalities

Why must denaturant be removed from the membrane prior to probe hybridization?
(A) To prevent nucleic acid and probe from forming hydrogen bonds prematurely
(B) To enhance probe binding
(C) To wash the membrane
(D) To sterilize the membrane

A. To prevent nucleic acid and probe from forming hydrogen bonds prematurely

What is the main use of Western blots?
(A) To detect specific proteins in mixtures using antibodies
(B) To detect DNA fragments
(C) To analyze RNA
(D) To perform PCR

A. To detect specific proteins in mixtures using antibodies

What is a probe in blotting analysis?
(A) A single-stranded nucleic acid fragment attached to a signal to identify specific DNA sequences
(B) An antibody that binds DNA
(C) A protein enzyme
(D) A dye for staining membranes

A. A single-stranded nucleic acid fragment attached to a signal to identify specific DNA sequences

What is Peptide Nucleic Acid (PNA)?
(A) DNA with unusual bases
(B) Modified nucleic acid with non-phosphodiester backbones
(C) RNA fragment in mitochondria
(D) Protein that binds DNA

B. Modified nucleic acid with non-phosphodiester backbones

Which blot technique confirms ELISA results?
(A) Southern blot
(B) Western blot, where patient serum is overlaid to detect antibodies binding viral proteins
(C) Northern blot
(D) Dot blot

B. Western blot, where patient serum is overlaid to detect antibodies binding viral proteins

How are DNA probes made?
(A) By cloning, restriction enzyme digestion, gel isolation
(B) By in vitro organic synthesis of nucleic acids
(C) By PCR
(D) All of the above

D. All of the above

What determines the specificity of DNA probes?
(A) Length of sequence — longer probes are more specific
(B) Color of probe
(C) Protein content
(D) Temperature alone

A. Length of sequence — longer probes are more specific

Do RNA/DNA or DNA/DNA probes bind more strongly?
(A) DNA/DNA
(B) RNA/DNA
(C) Both equally
(D) Neither bind strongly

B. RNA/DNA

What is the probe for Western blots?
(A) DNA
(B) RNA
(C) Antibodies
(D) Restriction enzymes

C. Antibodies

What are haptens?
(A) Complete antigens
(B) Incomplete antigens that can induce an immune response when linked to a carrier
(C) DNA fragments
(D) RNA molecules

B. Incomplete antigens that can induce an immune response when linked to a carrier

What are polyclonal antibodies?
(A) Antibodies from a single B-cell clone
(B) Antibodies produced by many B-cell clones responding to different epitopes on an antigen
(C) Antibodies that bind only one epitope
(D) Synthetic antibodies

B. Antibodies produced by many B-cell clones responding to different epitopes on an antigen

What are monoclonal antibodies?
(A) Antibodies produced by a single clone of B lymphocytes identical in structure and specificity
(B) Mixture of antibodies from various sources
(C) Antibodies with no specificity
(D) Antibodies binding all proteins

A. Antibodies produced by a single clone of B lymphocytes identical in structure and specificity

What are hybridomas?
(A) Hybrid cell lines formed by fusing antibody-producing B lymphocytes with myeloma cells, producing monoclonal antibodies
(B) Virus-infected cells
(C) Stem cells
(D) Bacterial colonies

A. Hybrid cell lines formed by fusing antibody-producing B lymphocytes with myeloma cells, producing monoclonal antibodies

What are two common nonradioactive tags used now for probes?
(A) Biotin and digoxigenin
(B) Fluorescein and rhodamine
(C) Radioactive phosphorus and sulfur
(D) ATP and GTP

A. Biotin and digoxigenin

What are three methods for adding tags to probes?
(A) End labeling, nick translation, random priming
(B) PCR, gel electrophoresis, Southern blot
(C) Heating, cooling, staining
(D) Cloning, sequencing, hybridization

A. End labeling, nick translation, random priming

What is specificity in testing?
(A) Percentage of times a test correctly identifies a positive result
(B) Percentage of times a test correctly identifies a negative result
(C) Percentage of false positives
(D) Percentage of false negatives

B. Percentage of times a test correctly identifies a negative result

What is stringency in hybridization?
(A) How well the probe and target bind; high stringency = high temp, low salt; low stringency = low temp, high salt
(B) Amount of probe used
(C) Length of probe only
(D) Color of probe

A. How well the probe and target bind; high stringency = high temp, low salt; low stringency = low temp, high salt

How are ideal conditions for probe hybridization calculated?
(A) By DNA concentration
(B) By melting temperature (Tm)
(C) By probe color
(D) By length of DNA fragment

B. By melting temperature (Tm)

What does Tm represent?
(A) The time it takes to hybridize DNA
(B) The amount of energy required to separate hybridized strands; at Tm half strands are denatured
(C) The temperature DNA melts in boiling water
(D) The length of DNA probe

B. The amount of energy required to separate hybridized strands; at Tm half strands are denatured

How does GC content affect stringency and Tm?
(A) Higher GC content increases stringency and increases Tm
(B) Lower GC content increases stringency
(C) GC content has no effect
(D) GC content lowers Tm

A. Higher GC content increases stringency and increases Tm

Tm for long probes is calculated by which formula?
(A) 81.5 + 16.6 log M + 0.41 (%G+C) - 0.61 (%formamide) - (600/n)
(B) 4 * (number of CG pairs) + 2 * (number of AT pairs)
(C) 100 - %GC content
(D) %AT pairs * 3

A. 81.5 + 16.6 log M + 0.41 (%G+C) - 0.61 (%formamide) - (600/n)

Tm for short probes is calculated by which formula?
(A) 81.5 + 16.6 log M + 0.41 (%G+C) - 0.61 (%formamide) - (600/n)
(B) 4°C × (number of CG pairs) + 2°C × (number of AT pairs)
(C) %GC content * 2
(D) %AT content * 4

B. 4°C × (number of CG pairs) + 2°C × (number of AT pairs)

What does Ct value represent?
(A) Sequence complexity or length of unique, non-repetitive nucleotide sequences in genome
(B) Concentration of probe
(C) Length of probe
(D) Time to hybridize

A. Sequence complexity or length of unique, non-repetitive nucleotide sequences in genome

How does hybridization buffer affect hybridization temperature?
(A) Increases optimal hybridization temperature
(B) Lowers optimal hybridization temperature
(C) No effect
(D) Denatures DNA

B. Lowers optimal hybridization temperature

What signals indicate successful probe detection?
(A) Chromogenic (color) and chemiluminescence (light)
(B) Radioactivity only
(C) Heat generation
(D) Sound emission

A. Chromogenic (color) and chemiluminescence (light)

Which two enzymes are used for probe signaling detection?
(A) Alkaline phosphatase and horseradish peroxidase
(B) DNA polymerase and RNA polymerase
(C) Ligase and helicase
(D) Cas9 and tracrRNA

A. Alkaline phosphatase and horseradish peroxidase

Nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate produce what signal when added to probe mixture?
(A) Blue color from dephosphorylation and redox reactions indicating probe binding
(B) Green fluorescence
(C) Radioactive emission
(D) No color change

A. Blue color from dephosphorylation and redox reactions indicating probe binding

What causes cross-hybridization bands and how to reduce them?
(A) Non-specific probe binding; increase stringency conditions
(B) Excess probe; dilute probe
(C) Wrong probe length; shorten probe
(D) Too little DNA; add more DNA

A. Non-specific probe binding; increase stringency conditions

What reaction does 1,2-dioxetane undergo with alkaline phosphatase and what signal is produced?
(A) Dephosphorylation; light (chemiluminescence)
(B) Phosphorylation; color
(C) Oxidation; heat
(D) Reduction; sound

A. Dephosphorylation; light (chemiluminescence)

Why is an internal control important for blot analysis interpretation?
(A) To determine expression relative to control and correct for errors in isolation, loading, and transfer
(B) To stain DNA
(C) To denature proteins
(D) To increase probe binding

A. To determine expression relative to control and correct for errors in isolation, loading, and transfer

What types of analysis can dot blots and slot blots be applied to?
(A) Gene expression, mutation, amplification, deletion
(B) DNA sequencing only
(C) Protein folding
(D) RNA splicing

A. Gene expression, mutation, amplification, deletion

What is a reverse dot blot?
(A) Probes immobilized on membrane, target labeled for hybridization
(B) Target immobilized on membrane, probes labeled
(C) Blotting DNA backwards
(D) Using RNA probes for DNA

A. Probes immobilized on membrane, target labeled for hybridization

What are macroarrays?
(A) Reverse dot blots best used with samples with large amounts of material, increasing number of targets assayed
(B) DNA sequencing chips
(C) Protein arrays
(D) Small dot blots

A. Reverse dot blots best used with samples with large amounts of material, increasing number of targets assayed

What is a microarray?
(A) Silicon or glass sheet with thousands of DNA probes to identify gene expression in tissue, ~80,000 spots
(B) Protein gel
(C) DNA sequencing machine
(D) A type of PCR

A. Silicon or glass sheet with thousands of DNA probes to identify gene expression in tissue, ~80,000 spots

How many genes are in the human genome approximately?
(A) 300
(B) 30,000
(C) 300,000
(D) 3,000

B. 30,000

Put these in order of complexity from most to least: proteome, genome, transcriptome
(A) Genome > Transcriptome > Proteome
(B) Proteome > Transcriptome > Genome
(C) Transcriptome > Proteome > Genome
(D) Genome > Proteome > Transcriptome

B. Proteome > Transcriptome > Genome

What are high density oligonucleotide arrays?
(A) Large numbers (>100,000) of probes synthesized directly on substrate via photolithography
(B) PCR machines
(C) Gel electrophoresis
(D) DNA sequencing tools

A. Large numbers (>100,000) of probes synthesized directly on substrate via photolithography

High density oligonucleotide arrays can be used to identify which mutations?
(A) Known mutations only
(B) Unknown mutations only
(C) Both known and unknown mutations
(D) Only frameshift mutations

C. Both known and unknown mutations

What are expression arrays used for?
(A) Reverse transcribe mRNA to cDNA, label with fluorescent dyes, hybridize to exon arrays, compare signal to measure gene expression
(B) Sequencing DNA
(C) Protein folding analysis
(D) Measuring DNA replication

A. Reverse transcribe mRNA to cDNA, label with fluorescent dyes, hybridize to exon arrays, compare signal to measure gene expression

What is comparative genomic hybridization?
(A) Molecular-cytogenetic method to determine copy number changes in DNA content, competitive assay with fluorophores on microarrays
(B) RNA expression analysis
(C) Protein sequencing
(D) DNA methylation analysis

A. Molecular-cytogenetic method to determine copy number changes in DNA content, competitive assay with fluorophores on microarrays

What is solution hybridization?
(A) Probe and target bind in solution, visualized on gel, used to measure mRNA expression
(B) DNA transferred to membrane
(C) Protein binding assay
(D) PCR technique

A. Probe and target bind in solution, visualized on gel, used to measure mRNA expression

What is gel mobility shift assay?
(A) Detects specific peptides by changes in electrophoretic migration speed upon antibody binding, identifies trans factors binding cis-acting elements
(B) DNA sequencing method
(C) Protein folding assay
(D) RNA degradation assay

A. Detects specific peptides by changes in electrophoretic migration speed upon antibody binding, identifies trans factors binding cis-acting elements

Name two ways to permanently bind nucleic acid to nitrocellulose following transfer.
(A) Baking at 80°C for 30 minutes and exposure to UV light for cross-linking
(B) Washing with water and staining
(C) Heating to 50°C and adding salt
(D) Using proteases

A. Baking at 80°C for 30 minutes and exposure to UV light for cross-linking

If a probe is dissolved in hybridization buffer with 50% formamide, is the stringency higher or lower than without formamide?
(A) Higher, because formamide facilitates DNA denaturation
(B) Lower, because formamide stabilizes DNA
(C) No change
(D) Formamide inhibits hybridization

A. Higher, because formamide facilitates DNA denaturation

If a high concentration of NaCl is added to hybridization solution, how is stringency affected?
(A) Stringency lowered, as salt promotes hydrogen bonding by bringing nucleic acids closer
(B) Stringency increased
(C) No effect
(D) DNA degraded

A. Stringency lowered, as salt promotes hydrogen bonding by bringing nucleic acids closer