MIC 102: Genetic Engineering
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78 Terms
Q: What is genetic engineering?
A: The deliberate manipulation of DNA to change an organism's genetic material.
Q: What is recombinant DNA?
A: DNA produced by combining DNA from different sources.
Recombinant DNA technology → Introduce, remove, or modify genes in an organism
Q: What is a cloning vector?
A: A DNA molecule used to carry foreign DNA into a host cell.
Most commonly used cloning vector → A plasmid
Q: What is a plasmid?
A: A small, circular, double-stranded DNA molecule that replicates independently of the bacterial chromosome.
Q: Are plasmids required for bacterial survival?
A: Usually no. They generally carry accessory genes that provide an advantage under certain conditions.
Q: Examples of genes commonly carried on plasmids.
A: Antibiotic resistance genes, virulence genes, and metabolic genes.
Q: Why are plasmids useful for genetic engineering?
A: They are easy to manipulate and can carry foreign DNA into bacterial cells.
Q: What feature allows plasmids to be copied independently of the bacterial chromosome?
A: Their own origin of replication (ori).
Q: What are restriction enzymes?
A: Enzymes that recognize specific DNA sequences and cut DNA at those sites.
“Restriction endonucleases”
Q: What type of DNA sequences do restriction enzymes recognize?
A: Specific recognition sequences, often palindromic (sequences that can be read the same forward/backwards)
Q: What is a palindromic DNA sequence?
A: A sequence that reads the same in the 5'→3' direction on both DNA strands.
Q: Why do bacteria naturally possess restriction enzymes?
A: To defend themselves against invading foreign DNA, such as bacteriophage DNA.
Q: How do bacteria protect their own DNA from their restriction enzymes?
A: Their DNA is methylated by restriction-modification systems.
DNA methyltransferase → Enzyme that methylates bacterial DNA
Q: What is the purpose of DNA methylation in restriction-modification systems?
A: It prevents restriction enzymes from cutting the bacterium's own DNA.
If NOT methylated, restriction enzyme would cut bacterium’s own chromosome
Q: What are sticky ends?
A: Short single-stranded DNA overhangs produced by some restriction enzymes.
A: Complementary sticky ends can base pair with one another, making DNA fragments easier to join.
Q: What are blunt ends?
A: DNA ends produced by straight cuts across both strands with no overhang.
Q: Which are generally easier to ligate: sticky ends or blunt ends?
Sticky ends → More efficient DNA fragments to be ligated by DNA ligase
Q: What bond does the enzyme DNA ligase form to join DNA fragments together?
A: A phosphodiester bond in the DNA backbone.
Q: After restriction digestion and ligation, what has been created?
A: Recombinant DNA.
Q: What three major components are needed to construct recombinant DNA?
Vector (usually a plasmid)
Foreign DNA fragment
DNA ligase
Q: What is the purpose of cutting both the plasmid and the foreign DNA with the same restriction enzyme?
A: To produce compatible DNA ends that can be joined together by DNA ligase.
Q: What are the two components of a restriction-modification system?
A: A restriction enzyme and a DNA methyltransferase.
Q: Which component destroys foreign DNA?
A: The restriction enzyme.
Q: Which component protects host DNA?
A: DNA methyltransferase.
Q: Why doesn't a bacterium digest (“cut”) its own chromosome?
A: Because its recognition sites are methylated.
Q: What type of DNA is primarily targeted by restriction enzymes?
A: Unmethylated foreign DNA.
Q: What is DNA cloning?
A: Producing many identical copies of a DNA fragment.
Q: What process introduces recombinant plasmids into bacteria?
A: Transformation → “Funeral pass”
Q: What is a selectable marker?
A: A gene that allows researchers to identify cells that received the plasmid.
An antiobiotic resistant gene is the most common selectable marker
Q: Why are antibiotic resistance genes included on cloning plasmids?
A: So only transformed bacteria survive on antibiotic-containing media.
Q: What happens to bacteria that fail to take up the plasmid when grown on selective media?
A: They die because they lack the resistance gene.
Transformed bacteria on selective media → Survive/form colonies
Q: Does growth on selective media prove that a bacterium contains the correct DNA insert?
A: No. It only shows that the bacterium contains the plasmid.
Q: What is the purpose of PCR?
Polymerase Chain Reaction → Amplify a specific DNA sequence
“Amplify” = Make millions of copies of a DNA fragment
Q: Is PCR performed inside living cells?
A: No. PCR is performed in vitro (in a test tube).
DNA polymerase carries out DNA synthesis during PCR
Q: Why is a heat-stable DNA polymerase required for PCR?
A: Taq DNA polymerase required, otherwise repeated heating would denature ordinary DNA polymerases.
Taq DNA polymerase is heat stable / survives repeated high temp.
Q: What are the five essential components of a PCR reaction?
A:
Template DNA
DNA polymerase (e.g., Taq DNA Polymerase)
Primers
dNTPs (ATP, GTP, etc)
Buffer containing Mg²⁺ (Essential cofactor for DNA polymerase activity)
Q: What are primers in PCR reaction?
A: Short single-stranded DNA molecules that define the region to be amplified and provide a starting point for DNA polymerase.
Two required — One forward primer and one reverse primer
Q: Why can't DNA polymerase begin DNA synthesis without primers?
A: DNA polymerase can only extend an existing 3′ end; it cannot start DNA synthesis de novo.
Q: Which PCR component determines the exact DNA region that will be amplified?
A: The primers.
Q: What are the three major steps of one PCR cycle?
Denaturation (92C)
Annealing (approx 60C)
Extension (72C)
Q: What happens during the denaturation step?
A: 92C → High temperature separates the two DNA strands by breaking hydrogen bonds.
Q: What happens during the annealing step?
A: 50-65C → Primers bind (hybridize) to complementary DNA sequences.
Q: What happens during the extension step?
A: 72C → DNA polymerase synthesizes new DNA beginning at each primer.
Q: During which PCR step does DNA polymerase actually synthesize DNA?
A: Extension
Q: During which PCR step do primers bind to the template?
Annealing
Q: During which PCR step are the DNA strands separated?
A: Denaturation.
Q: Why is PCR called a "chain reaction"?
A: Newly synthesized DNA molecules become templates in the next cycle, causing exponential amplification.
Q: Does PCR increase DNA linearly or exponentially?
A: Exponentially.
Q: What are common applications of PCR?
A: DNA cloning, pathogen detection, genetic engineering, DNA sequencing, and forensic analysis.
Q: Why is PCR useful before cloning a gene?
A: It generates many copies of the DNA fragment to be inserted into a vector.
Q: Can PCR amplify an entire genome at once?
A: No. PCR amplifies only the DNA sequence located between the two primers.
Q: Which PCR reagent determines the beginning and end of the amplified DNA fragment?
A: The primers.
Q: Which PCR reagent actually synthesizes the new DNA strand?
A: Taq DNA polymerase.
Q: What would happen if ordinary DNA polymerase were used instead of Taq polymerase?
A: It would become denatured during the high-temperature denaturation step.
Q: What is gel electrophoresis?
A: A technique used to separate DNA fragments according to size.
Q: What property of DNA allows it to move through an electric field?
A: DNA has a negatively charged phosphate backbone.
Q: Toward which electrode does DNA migrate during gel electrophoresis?
A: The positive electrode (anode).
Q: Which DNA fragments migrate farther through the gel?
A: Smaller DNA fragments → Experience less resistance moving through pores of the gel
Q: What is the purpose of an agarose gel?
A: It acts as a molecular sieve that separates DNA fragments by size.
Q: What is a DNA ladder (DNA marker)?
A: A mixture of DNA fragments of known sizes used to estimate the sizes of unknown DNA fragments.
Q: What is the general order of a bacterial cloning experiment?
Obtain plasmid
Cut DNA with restriction enzyme
Cut plasmid with the same restriction enzyme
Ligate insert into plasmid
Transform bacteria
Select transformed cells
Grow colonies
Q: What is the primary purpose of PCR?
A: To amplify (make many copies of) a specific DNA sequence.
Q: What is the primary purpose of gel electrophoresis?
A: To separate DNA fragments based on size.
Q: What is selective media?
A: Growth media containing an agent (such as an antibiotic) that allows only certain bacteria to grow.
Q: What characteristic allows transformed bacteria to survive on selective media?
A: They carry the plasmid's antibiotic resistance gene.
Q: Match each technique with its primary purpose:
PCR
Restriction enzymes
DNA ligase
Gel electrophoresis
PCR → Amplify DNA
Restriction enzymes → Cut DNA
DNA ligase → Join DNA
Gel electrophoresis → Separate DNA fragments by size
Q: What does CRISPR stand for?
A: Clustered Regularly Interspaced Short Palindromic Repeats.
Q: What is the normal biological function of the CRISPR-Cas system?
A: It serves as an adaptive immune system that protects bacteria from invading viruses (bacteriophages).
Q: Which enzyme performs the DNA cutting in the CRISPR-Cas9 system?
A: Cas9
Q: What determines where Cas9 cuts DNA?
A: A guide RNA (gRNA) directs Cas9 to a complementary DNA sequence
Provides sequence specificity by base-pairing w/ target DNA
Q: Why is CRISPR-Cas9 considered programmable?
A: Changing the guide RNA changes the DNA sequence that Cas9 targets.
Q: What type of DNA break does Cas9 typically create?
A: A double-stranded DNA break → Cell’s DNA repair mechanisms can be used to disrupt/modify genes after the break
Q: Which system allows targeted genome editing?
A: CRISPR-Cas9.
Q: Match each molecular tool with its primary function.
Tool | |
|---|---|
Restriction enzyme | |
DNA ligase | |
PCR | |
Gel electrophoresis | |
CRISPR-Cas9 |
Function |
|---|
Cut DNA |
Join DNA |
Amplify DNA |
Separate DNA |
Edit DNA |
Place these cloning steps in the correct order.
Ligation
PCR
Transformation
Restriction digestion
Selection
PCR (if amplification is needed)
Restriction digestion
Ligation
Transformation
Selection
Q: Which natural bacterial process is exploited during molecular cloning?
Transformation
Q: Which step actually creates recombinant DNA?
A: DNA ligase joining the insert to the plasmid.
Q: Which step identifies bacteria that successfully received the plasmid?
A: Selection on antibiotic-containing media.