Molecular Genetics: Mutations and Genetic Engineering

Comprehensive practice flashcards covering mutation mechanisms, phenotypic outcomes, repair pathways, and recombinant DNA engineering technologies.

Mutation

A heritable alteration in the DNA sequence of an organism, encompassing both the biological process by which sequence change occurs and the resulting altered genetic product.

Forward Genetics

The classical genetic strategy that begins with an identified mutant phenotype and works toward determining the underlying genetic sequence or genotype.

Reverse Genetics

A modern genetic approach that alters or mutates a specific known DNA sequence or gene to investigate its resulting phenotypic consequence.

Antennapedia Mutation

A homeotic mutation in Drosophila melanogaster that alters developmental identity, causing adult legs to form on the head where antennae normally develop.

Indy Gene

A gene in Drosophila melanogaster encoding an intermediate transporter of the Krebs cycle; its name stands for 'I\'m Not Dead Yet' because mutated flies reportedly exhibit extended longevity.

$\Delta\text{F508}$ Mutation

The most common mutation causing cystic fibrosis, consisting of a codon deletion ($\Delta$) that eliminates phenylalanine ($\text{F}$) at amino acid position $508$ in the CFTR protein.

Sickle-Cell Anemia ($\text{E6V}$) Mutation

A single nucleotide polymorphism (SNP) from $\text{GAG}$ to $\text{GTG}$ in the $\beta$-globin gene that substitutes glutamic acid ($\text{E}$) with valine ($\text{V}$) at position $6$, producing abnormal hemoglobin S ($\text{HbS}$).

$\text{CCR5}\Delta 32$ Mutation

A $32\,\text{bp}$ deletion in the open reading frame of the CCR5 coreceptor gene that prevents HIV-1 cellular entry and is associated with historical protection against plague and smallpox.

Penicillin-Binding Proteins (PBPs)

Bacterial enzymes involved in peptidoglycan cell wall biosynthesis that serve as targets for $\beta$-lactam antibiotics; point mutations in these proteins confer penicillin resistance.

Omicron Variant (SARS-CoV-2)

A viral variant emerging in late $2021$ with over $50$ mutations ($32$ located in the spike protein), associated with high viral replication in the upper respiratory tract, lower load in the lungs, and elevated immune escape.

Sickle Cell Trait

A heterozygous state carrying one normal allele and one $\beta$-globin $\text{E6V}$ allele ($\text{HbA}/\text{HbS}$), providing a selective advantage against severe malaria without developing full sickle-cell disease.

Transition Mutation

A point mutation that substitutes a purine for another purine ($\text{A} \leftrightarrow \text{G}$) or a pyrimidine for another pyrimidine ($\text{C} \leftrightarrow \text{T}$).

Transversion Mutation

A point mutation in which a purine is replaced by a pyrimidine, or a pyrimidine is replaced by a purine ($\text{A}$ or $\text{G} \leftrightarrow \text{C}$ or $\text{T}$).

Silent Mutation

A nucleotide substitution in a protein-coding sequence that alters a codon but does not change the specified amino acid due to genetic code degeneracy.

Missense Mutation

A base substitution that changes an amino acid codon to code for a different amino acid, yielding effects ranging from neutral to total loss of protein function.

Nonsense Mutation

A base substitution that converts an amino acid-specifying codon into a premature stop codon, generating a truncated and typically nonfunctional polypeptide.

Frameshift Mutation

The insertion or deletion of a number of nucleotides that is not a multiple of three, altering the translational reading frame downstream of the mutation site.

Conditional Mutation

A mutation whose phenotypic expression is dependent upon specific environmental parameters, such as elevated or reduced temperatures.

Spontaneous Deamination

The hydrolytic removal of an amino group from a base, such as the conversion of cytosine to uracil, causing a subsequent $\text{GC} \rightarrow \text{AT}$ transition if left unrepaired.

Spontaneous Depurination

The breakage of the N-glycosidic bond releasing a purine (adenine or guanine) and leaving an apurinic (AP) site, occurring up to $5{,}000$ times daily per human cell.

Nucleoside Analogs

Chemical mutagens structurally similar to normal nitrogenous bases (e.g., $2$-aminopurine, $5$-bromouracil) that are mistakenly incorporated during replication and promote mispairing.

Intercalating Agents

Chemical mutagens (e.g., ethidium bromide, acridine orange) that wedge between adjacent base pairs, distorting helix conformation and inducing replication slippage that leads to frameshifts.

Thymine Dimers

Intrastrand covalent photoproducts formed between adjacent thymine residues upon exposure to non-ionizing ultraviolet (UV) radiation, obstructing replication and transcription.

DNA Polymerase Proofreading

An intrinsic $3' \rightarrow 5'$ exonuclease activity by which DNA polymerase detects mispaired terminal bases during synthesis, excises them, and resumes elongation with the correct base.

Methyl-Directed Mismatch Repair

A post-replication repair system that relies on temporary hemimethylation of newly synthesized DNA, utilizing MutS, MutL, and MutH to identify, excise, and repair mismatches on the unmethylated strand.

Nucleotide Excision Repair (NER)

A dark repair pathway in which a multi-protein nuclease cuts both sides of a bulky DNA lesion (e.g., a thymine dimer), helicase removes the excised segment, DNA polymerase I fills the gap, and ligase seals the nick.

Photoreactivation (Light Repair)

A direct repair process in bacteria and plants (absent in mammals) where the photolyase enzyme uses energy absorbed from visible light to break the covalent bonds of pyrimidine dimers.

Base Excision Repair (BER)

A repair pathway that removes modified or damaged bases (such as uracil or apurinic sites) using AP endonucleases to nick the damaged phosphodiester backbone prior to gap filling and ligation.

Replica Plating

A screening technique that transfers bacterial colonies via a sterile velvet surface onto nutrient-deficient versus complete media to identify auxotrophic mutants unable to synthesize essential metabolites.

Ames Test

A bacterial biological assay that measures the rate of reversion of histidine-auxotrophic Salmonella typhimurium to prototrophy in the presence of rat liver extract to screen chemicals for mutagenicity and carcinogenicity.

Recombinant DNA Technology

The collection of laboratory techniques used to cut, join, and manipulate DNA sequences in vitro, creating novel recombinant DNA molecules that combine genetic material from multiple sources.

Transgenic Organism

An organism that contains foreign, recombinant DNA engineered in vitro and introduced into its genome.

Polymerase Chain Reaction (PCR)

A cyclical enzymatic process amplifying specific target DNA sequences in vitro via successive steps of thermal denaturation ($95\,^\circ\text{C}$), primer annealing ($\sim 50\,^\circ\text{C}$), and polymerase extension ($72\,^\circ\text{C}$).

Taq Polymerase

A heat-stable DNA polymerase purified from the thermophilic bacterium Thermus aquaticus, retaining functionality during PCR thermal cycling with optimal activity near $70\,^\circ\text{C}$.

Bacteriophage T7 RNA Polymerase

A viral RNA polymerase with high specificity for its cognate promoter, widely utilized in recombinant genetic procedures for in vitro transcription.

Agarose Gel Electrophoresis

A separation method in which charged DNA fragments migrate through a porous agarose gel matrix under an electric field, separating linearly based on molecular size toward the positive anode.

DNA Ladder

A standardized mixture of double-stranded DNA fragments of known base-pair lengths loaded onto an agarose gel to calibrate the molecular weight of experimental DNA samples.

SYBR Green

A fluorescent dye that selectively intercalates into double-stranded DNA, commonly used to visualize DNA bands in electrophoresis or quantify amplification during real-time PCR.

Restriction Endonucleases

Bacterial defense enzymes that cleave foreign viral DNA at specific palindromic recognition sequences, typically $4$ to $8\,\text{bp}$ long, yielding sticky or blunt ends.

EcoRI

A restriction endonuclease from Escherichia coli that cleaves the palindromic motif $5'\text{-GAATTC-}3'$, producing staggered cuts with complementary $5'$ sticky overhangs.

Sticky Ends

Staggered, single-stranded nucleotide overhangs generated by asymmetric cleavage by restriction enzymes, allowing spontaneous base pairing with complementary overhangs.

Blunt Ends

Termini of cleaved DNA molecules lacking single-stranded overhangs, formed when restriction enzymes (e.g., HaeIII) cleave directly across both strands at the same central phosphodiester bond.

DNA Ligase

An enzyme catalyzing the condensation between a terminal $5'$ phosphate and a neighboring $3'$ hydroxyl group on double-stranded DNA, utilizing ATP and releasing AMP to paste fragments together.

Alkaline Phosphatase (Cloning)

An enzyme used in recombinant cloning to remove the terminal $5'$ phosphate groups from digested vector DNA, preventing self-ligation and recircularization of the vector backbone.

Multiple Cloning Site (MCS)

A short, synthetically engineered tract within a plasmid vector containing unique recognition sites for numerous restriction endonucleases to facilitate targeted insert ligation.

Recombinant Human Insulin Production

The biotechnological process of inserting the human insulin gene into a plasmid vector and expressing it in recombinant bacteria to secrete authentic human insulin.

Genomic Library

A comprehensive repository of recombinant host cells (usually bacteria) containing plasmid or viral vectors that together represent the entire fragmented genome of an organism.

Dideoxynucleotide (ddNTP)

A modified nucleotide lacking both $2'$ and $3'$ hydroxyl groups on the ribose ring, which halts phosphodiester chain elongation once incorporated by DNA polymerase.

Sanger Sequencing

A classic DNA sequencing method utilizing four fluorochrome-labeled chain-terminating ddNTPs, DNA polymerase, and capillary gel electrophoresis to determine DNA sequences up to $\sim 1000\,\text{bp}$.

Quantitative PCR (qPCR)

A real-time fluorometric amplification procedure where the accumulation of double-stranded DNA is tracked cycle-by-cycle to quantify the absolute or relative abundance of starting target molecules.

Reverse Transcriptase PCR (RT-PCR)

A method using viral reverse transcriptase to synthesize complementary DNA (cDNA) from mature RNA templates, followed by PCR amplification to assess gene expression without genomic introns.

Synthetic Biology

An engineering discipline that designs, standardizes, and stitches modular biological parts (promoters, ORFs, operators, terminators) into artificial genetic circuits.

Complementary DNA (cDNA)

Double-stranded DNA generated in vitro from eukaryotic messenger RNA by reverse transcriptase, representing the uninterrupted exonic sequence without introns.

Eukaryotic Shuttle Vector

A specialized cloning plasmid designed to replicate in both bacteria and eukaryotic cells (e.g., pEGFP-N1), carrying bacterial origins and selection markers alongside eukaryotic promoters and polyadenylation signals.

Electroporation

A physical method of introducing foreign nucleic acids into host cells by applying brief, high-intensity electrical pulses that transiently permeabilize the phospholipid membrane.

Microinjection

A physical delivery technique that uses an ultra-fine glass micropipette to inject recombinant DNA directly into the nucleus or cytoplasm of a single eukaryotic cell or embryo.

Transcriptional Gene Fusion

A recombinant construct joining the promoter of a target gene to an independent reporter gene, allowing quantification of promoter activity and tissue-specific transcription without altering the protein of interest.

Translational Gene Fusion

A genetic construct fusing the coding sequence of a gene of interest in-frame with a reporter tag via a linker peptide, generating a hybrid chimeric protein to visualize subcellular localization.

Reporter Genes

Selectable or visual markers—including green fluorescent protein (GFP), $\beta$-galactosidase (lacZ), and luciferase—whose observable products indicate whether, where, and when a fused gene is expressed.

CRISPR-Cas9 System

An adaptive immune mechanism from Streptococcus pyogenes engineered for targeted genome editing, combining an RNA guide with Cas9 endonuclease to generate site-specific double-strand breaks repaired by NHEJ or HDR.