Recombinant DNA Technology Flashcards

Question

What are the requirements for PCR?

Answer 1

Template DNA (with known flanking sequences) Primers (forward + reverse) Taq DNA polymerase (heat-resistant) dNTPs Buffer with Mg²⁺ Thermal cycler

Answer 2

Denaturation (95°C) – DNA strands separate. Annealing (40–65°C) – Primers bind to target sequences. Extension (72°C) – Taq polymerase synthesizes new DNA strands. 🔁 Repeated for 20–40 cycles → Exponential amplification (2ⁿ copies).

Answer 3

Short DNA oligonucleotides (17–25 nucleotides). Define specificity of PCR. Can be engineered to: Introduce restriction sites Insert tags for protein purification Introduce mutations Add flanking regions for recombination

Answer 4

Matches 5’ end of top strand

Answer 5

Reverse primer is complementary to 3’ end of top strand (i.e., 5’ end of bottom strand).

Answer 6

Taq: Fast but error-prone (285 errors per 10⁶ bases). Pfu: High fidelity with proofreading ability (3’–5’ exonuclease activity).

Answer 7

Separates DNA/RNA fragments by size. Smaller fragments move faster through the agarose gel. DNA migrates toward positive electrode (because DNA is negatively charged). Can visualize DNA using: Ethidium bromide (EtBr) SYBR Safe (safer alternative) DNA ladder is used to estimate fragment sizes.

Answer 8

Recombinant DNA can be inserted into expression vectors to produce proteins. Advantages: Fast growth Easy manipulation Scalable for industrial production

Answer 9

Introns: Bacteria can’t process them → use cDNA, not genomic DNA. Promoters/terminators: Eukaryotic ones don’t work in bacteria → use bacterial expression vectors. Ribosome binding sites (RBS): Eukaryotic mRNA lacks bacterial RBS → vectors must include one.

Answer 10

Insulin – E. coli Growth hormone – E. coli Chymosin – E. coli, yeast, fungi Hepatitis B vaccine – Yeast Herceptin (antibody) – Mammalian cells Antithrombin III – Transgenic goats

Answer 11

Clone gene into a plasmid vector with bacterial promoter. Transform into E. coli. Select colonies (e.g., using ampicillin resistance). Induce expression. Lyse cells. Purify protein.

Answer 12

Modern labs can order a synthetic gene from companies. Entire gene (~1.5 kb) can be delivered in <1 month. Useful for: Adding/removing restriction sites Codon optimization Avoiding need for a DNA source

Answer 13

Goal: Produce RNA in a lab environment using a DNA template. How: Use plasmid vectors (like pGEM3Z) that contain promoters for T7 or SP6 RNA polymerases, which allow in vitro transcription.

Answer 14

Insert the gene for SARS-CoV-2 spike protein into the plasmid. Transcribe RNA in vitro using modified nucleotides (e.g., pseudouridine instead of uridine for stability and reduced immune reaction). Add a 5’ cap and 3’ poly-A tail to mimic eukaryotic mRNA. Remove DNA template, purify the mRNA. Package it into lipid nanoparticles → vaccine ready for injection

Answer 15

STRs (Short Tandem Repeats): Short DNA sequences (2–6 base pairs) repeated many times (e.g., TCAT). Individuals vary in the number of repeats → basis for DNA profiling. Use PCR primers that flank the repeat regions → amplify the STRs. PCR product size = number of repeats → compare between individuals.

Answer 16

Forensics: Analyze >10 STR regions to match DNA from crime scenes to suspects. Identical STR profiles are nearly unique to individuals (except identical twins). Paternity Testing: A child inherits one STR allele from each parent.

Answer 17

Use nucleic acid probes that bind (hybridize) to complementary sequences. Detection methods involve blotting techniques: Southern blot: DNA Northern blot: RNA Western blot: Protein These methods separate molecules by gel electrophoresis, transfer to a membrane, then detect with a labeled probe.

Answer 18

Measures mRNA levels, indicating gene expression. Shows when and where a gene is active. Example: In fish development, detect when the troponin-T gene is expressed.

Answer 19

Measures DNA amplification in real-time, using fluorescent dyes or probes. SYBR Green: binds any double-stranded DNA. TaqMan probe: sequence-specific probe that fluoresces when degraded.

Answer 20

Number of PCR cycles required to reach detectable fluorescence. Lower Ct = more starting template.

Answer 21

qPCR: Quantifies DNA. qRT-PCR: Reverse transcribes RNA to cDNA, then quantifies → used for RNA viruses and gene expression.

Answer 22

Extract RNA from patient sample → convert to cDNA → amplify genes like S, N, ORF1ab. Use controls to validate: Negative control: ensures no contamination. Positive control: confirms RT and PCR worked. Internal control: confirms RNA was successfully extracted. Variant detection: Some variants (e.g., Omicron) have deletions in the S gene → absence of S signal but presence of N and ORF1ab = variant present

Answer 23

Sequencing single genes or PCR fragments. Used to: Check cloned DNA sequences. Identify known mutations.

Answer 24

Sequences entire genomes or transcriptomes. Applications: Whole-genome sequencing. Transcriptomics (gene expression). Prenatal diagnosis, disease gene discovery, and microbiome studies (metagenomics).

Answer 25

A transgenic animal has foreign (exogenous) DNA inserted into its genome

Answer 26

To produce useful proteins in milk (e.g. for medical use). To improve farm animal traits (e.g. growth, nutrition). To study genes for research (model organisms).

Answer 27

DNA is injected into a fertilized egg (zygote), directly into one of the pronuclei. Typically, 1–200 copies of the DNA insert into the genome at one location. Used in various animals including mice, goats, and fish.

Answer 28

A genetically modified salmon with: A growth hormone gene from Chinook salmon. A promoter from ocean pout, allowing year-round expression.

Answer 29

Grows faster. More efficient feed use.

Answer 30

Health risks (e.g. allergens or hormone levels). Environmental impact if they escape and breed with wild fish. ✅ Approved for sale in Canada and the USA

Answer 31

Goats are engineered to secrete human proteins (e.g. antithrombin, used to prevent blood clots) in their milk. This is a cost-effective way to produce therapeutic proteins.

Answer 32

Produce proteins for industry/pharma. Improve crops (resistance to pests, herbicides, better nutrition).

Answer 33

Agrobacterium tumefaciens is a natural plant pathogen that causes crown gall tumors. It uses a Ti (tumor-inducing) plasmid, which integrates its T-DNA into plant genomes.

Answer 34

Scientists replace T-region genes with genes of interest. The modified plasmid is used to infect plant cells. These cells are then regenerated into whole transgenic plants.

Answer 35

Bt Corn/Cotton: Contains a gene from Bacillus thuringiensis that produces an insecticidal protein. Herbicide-Resistant Soy: Normal soy plants are killed by glyphosate (a herbicide). Transgenic soy contains a bacterial EPSPS enzyme that is not affected by glyphosate. Adoption: Cotton: 80% GM Soybean: 77% GM Maize: 32% GM → Economic gain: ~$186 billion (1996–2016)

Answer 36

Could spread resistance to weeds ("superweeds"). Overreliance on a single herbicide. Unknown health/environmental effects.

Answer 37

CRISPR: A bacterial immune system that keeps memory of viruses using DNA fragments stored in CRISPR arrays. Cas9: An enzyme that cuts DNA at locations specified by guide RNAs.

Answer 38

After viral infection, bacteria store viral DNA in their genome (CRISPR locus). When the virus infects again, crRNA + tracrRNA + Cas9 recognize and destroy it.

Answer 39

Engineered sgRNA (single guide RNA) guides Cas9 to any gene of interest. Cas9 makes a double-strand break at the target location.

Answer 40

NHEJ (Non-Homologous End Joining): Fast but error-prone. Leads to small insertions/deletions → can knock out a gene. HDR (Homology-Directed Repair): Accurate, uses a template to repair DNA. Can edit genes precisely (e.g., correct mutations, insert new sequences).

Answer 41

Yeast (Saccharomyces cerevisiae): Delete the ADE2 gene → red colonies (due to a metabolic block). Restore function using gene from another species via HDR. CAR-T Cell Therapy: Modify patient T cells using CRISPR to fight cancer more effectively.

Recombinant DNA Technology Flashcards

(65 cards)