BIO 111 – Chapter 11 DNA Technology – Vocabulary Flashcards

Genetic Engineering, Biotechnology & Foundational Vocabulary

• Genetic Engineering (GE): Direct DNA manipulation to predictably change phenotype, often using recombinant-DNA (rDNA) tools and vectors.

• Biotechnology: Broader field involving commercial/research use of organisms or bioprocesses (GE is a sub-discipline).

• Recombinant DNA (rDNA): DNA from two+ sources, created with restriction enzymes and DNA ligase.

• Transgenic Organism (GMO): Carries foreign rDNA, transmissible through germ line.

• cDNA: Double-stranded DNA copy from mRNA, made by reverse transcriptase; lacks introns, ideal for bacterial expression.

• Central Enzymes: DNA → RNA uses RNA polymerase; RNA → DNA uses Reverse transcriptase.

Vectors & Plasmids

• Plasmid: Small, circular, self-replicating bacterial DNA, often carrying antibiotic resistance.

• Role in GE: Serve as cloning vectors to carry, replicate, and express foreign DNA in host cells.

• Alternate Vectors: Bacteriophages, cosmids, BACs, YACs, viral vectors.

Restriction Enzymes & DNA Ligation

• Restriction Enzyme: Cuts both DNA strands at specific palindromic restriction sites (4–8 bp).

• Sticky vs. Blunt Ends: Offset cuts create sticky ends (overhangs for hydrogen bonding); straight cuts create blunt ends (no overhang).

• DNA Ligase: Forms ext{phosphodiester} bonds, joining vector and insert permanently.

Recombinant Human Insulin Example

• Human insulin gene cloned into bacterial plasmid; universal genetic code allows bacteria to produce identical human insulin.

Sanger (Chain-Termination) Gene Sequencing

• Uses a primer, normal dNTPs, and terminator ddNTPs (lacking 3'-OH, fluorescently labeled) with DNA Polymerase to synthesize strands. Fragments are separated by capillary electrophoresis, and fluorescent tags are read to determine the DNA sequence.

Coding vs. Non-Coding DNA

• Coding DNA (Exons): Translated into protein.

• Non-Coding DNA: Includes introns, promoters, enhancers, repetitive elements; regulatory or no known function. Only 1 ext{–}2\% of human genome encodes proteins.

Polymerase Chain Reaction (PCR)

• Definition: In-vitro technique to amplify a chosen DNA region exponentially (2^n copies per cycle).

• Denature DNA: Separate double helix by heat (≈ 94!^{\circ}\text{C}).

• Enzyme: Taq polymerase (thermostable).

DNA Profiling (DNA Fingerprinting)

• STRs (Short Tandem Repeats): 2–6 bp units repeated; highly polymorphic, in non-coding regions, excellent individual markers.

Gel Electrophoresis

• Purpose: Separates DNA (or RNA/proteins) by size.

• Gel Matrix: Agarose (large fragments) or polyacrylamide (small).

• Migration Rule: Smaller fragments move farther/faster toward the positive electrode; DNA’s phosphate backbone is negatively charged.

• Single vs. Double Band: Homozygous for fragment length → one band; Heterozygous → two bands.

Stem Cells

• General Definition: Undifferentiated cells capable of self-renewal and differentiation into specialized types.

• Totipotent: Can form all embryonic + extra-embryonic tissues (zygote).

• Pluripotent: Can form all body cell types but NOT placenta (Embryonic Stem Cells).

• Adult (Multipotent) Stem Cells: Restricted lineages.

Somatic Cell Nuclear Transfer (SCNT) & Dolly

• Participants: Mammary gland donor (nucleus), egg donor (denucleated egg), surrogate mother.

• Denucleated Egg: Oocyte with nucleus removed; retains cytoplasm + mitochondria.

• Are Clones Identical?: Nuclear genome identical to donor, but mitochondrial DNA from egg donor; epigenetic marks + environment cause phenotypic differences.

Tissue Cloning (Therapeutic Cloning)

• Creating genetically matched tissues/organs to avoid immune rejection by differentiating SCNT-derived cells or reprogramming somatic cells.

DNA Probes

• Single-stranded, labeled DNA/RNA fragment that hybridizes to a complementary target, used in various blotting techniques.

Genetic Testing Time-Points

• Preconception: Carrier screening in parents.

• Preimplantation: PGD/PGT of IVF embryos before transfer.

• Post-Implantation (Prenatal): CVS, amniocentesis, cell-free fetal DNA in utero.

• Post-Delivery (Newborn): Heel-prick panel for metabolic disorders, SCID.

• The terms describe settings for biological processes:

  • In vivo: Within a living organism.

  • In vitro: Outside a living organism, typically in a controlled environment (e.g., test tube). PCR is an in-vitro technique.

  • In utero: Within the uterus; refers to prenatal conditions or testing.

Gene Therapy & Genome Editing

• Goal: Treat/cure disease by adding, removing, or correcting genes.

• Gene Silencing: Knockdown/knockout expression (e.g., siRNA, CRISPR interference).

• Gene Editing: Physically changes DNA sequence (insert, delete, substitute).

• CRISPR/Cas9: Guide RNA directs Cas9 nuclease to target; causes double-strand break, repaired by NHEJ (indels) or HDR (precise edits).

• Prime Editor: Cas9 nickase + reverse transcriptase + pegRNA; introduces precise edits WITHOUT double-strand break or donor DNA.

Vaccines—Traditional vs. mRNA

• Traditional: Delivers whole (killed/weakened) virus or purified antigen to stimulate immunity (memory B & T cells).

• mRNA Vaccines: Lipid-nanoparticle–encapsulated mRNA encoding viral spike protein; host ribosomes translate mRNA to antigen, triggering immune response.

• Advantages: Rapid design, no live pathogen, easily reprogrammed, strong immunity.

• Disadvantages: Requires ultra-cold storage, mRNA less stable, potential short-term reactogenicity.

Genetic Engineering, Biotechnology & Foundational Vocabulary

• Genetic Engineering (GE): Direct DNA manipulation, often via recombinant-DNA (rDNA).

• Biotechnology: Broader field using organisms/bioprocesses; GE is a sub-discipline.

• Recombinant DNA (rDNA): DNA from 2+ sources, joined by restriction enzymes and DNA ligase.

• Transgenic Organism (GMO): Carries foreign rDNA.

• cDNA: Double-stranded DNA from mRNA, lacks introns.

• Central Enzymes: RNA polymerase (DNA \to RNA); Reverse transcriptase (RNA \to DNA).

Vectors & Plasmids

• Plasmid: Small, circular bacterial DNA, used as cloning vectors to carry foreign DNA.

Restriction Enzymes & DNA Ligation

• Restriction Enzyme: Cuts DNA at specific restriction sites, creating sticky or blunt ends.

• DNA Ligase: Connects DNA fragments permanently.

Recombinant Human Insulin Example

• Human insulin gene cloned into bacterial plasmid, allowing bacterial production.

Sanger (Chain-Termination) Gene Sequencing

• Uses primer, normal dNTPs, and fluorescently labeled terminator ddNTPs with DNA Polymerase to sequence DNA fragments.

Coding vs. Non-Coding DNA

• Coding DNA (Exons): Translated into protein (1 ext{–}2\% of human genome).

• Non-Coding DNA: Regulatory or no known function (e.g., introns, promoters).

Polymerase Chain Reaction (PCR)

• Definition: In-vitro technique to exponentially amplify specific DNA regions (2^n copies).

• Enzyme: Taq polymerase (thermostable).

DNA Profiling (DNA Fingerprinting)

• STRs (Short Tandem Repeats): Highly polymorphic units in non-coding regions, used as individual markers.

Gel Electrophoresis

• Purpose: Separates DNA by size; smaller fragments move farther/faster towards the positive electrode.

Stem Cells

• General Definition: Undifferentiated cells capable of self-renewal and differentiation.

• Types: Totipotent (all embryonic + extra-embryonic), Pluripotent (all body cells), Adult (Multipotent) (restricted lineages).

Somatic Cell Nuclear Transfer (SCNT) & Dolly

• Process: Nuclear transfer from somatic cell into denucleated egg, creating a clone with nuclear DNA from donor and mitochondrial DNA from egg donor.

Tissue Cloning (Therapeutic Cloning)

• Creates genetically matched tissues/organs to avoid immune rejection.

DNA Probes

• Labeled single-stranded DNA/RNA fragments that hybridize to complementary targets.

Genetic Testing Time-Points

• Stages: Preconception, Preimplantation, Post-Implantation (Prenatal), Post-Delivery (Newborn).

• Context terms: In vivo (within organism), In vitro (outside organism), In utero (within uterus).

Gene Therapy & Genome Editing

• Goal: Treat/cure disease by modifying genes.

• Gene Silencing: Reduces/eliminates gene expression (e.g., siRNA, CRISPRi).

• Gene Editing: Physically alters DNA sequence (insert, delete, substitute).

• CRISPR/Cas9: Uses guide RNA to target Cas9 nuclease for DNA double-strand breaks.

• Prime Editor: Introduces precise edits without double-strand breaks.

Vaccines—Traditional vs. mRNA

• Traditional: Delivers whole/weakened virus or antigen for immune stimulation.

• mRNA Vaccines: mRNA encoding viral protein (e.g., spike) is delivered, host cells translate it to trigger an immune response.

• mRNA Advantages: Rapid design, no live pathogen, strong immunity. Disadvantages: Ultra-cold storage, less stable.