Molecular Biology Technologies, Cell Studies, and Systems Biology

Comprehensive vocabulary flashcards covering DNA manipulation, cloning, sequencing technologies, protein analysis methods, structural biology, and systems biology principles.

Restriction Endonuclease

A bacterial enzyme that recognizes and cleaves double-stranded DNA at specific palindromic sequences (typically $4\text{--}8\text{ bp}$), originally functioning in nature as a bacterial immune defense against foreign bacteriophage DNA.

EcoRI Recognition Site

The specific 6-base pair palindromic sequence $5'\text{-GAATTC-}3'$ cleaved by the EcoRI endonuclease between G and A, generating staggered sticky ends with $5'$ single-stranded overhangs ($5'\text{-AATT-}3'$).

Agarose Gel Electrophoresis

A technique that uses an electric current to separate charged nucleic acid fragments through an agarose mesh, causing negatively charged DNA to migrate toward the positive electrode at a speed inversely proportional to fragment size.

Restriction Mapping

A method used to determine the locations of restriction enzyme cut sites along a DNA sequence by digesting DNA samples with single and double enzyme combinations and measuring fragment lengths via gel electrophoresis.

pBR322

A standard $4361\text{ bp}$ bacterial plasmid cloning vector engineered with an origin of replication ($\text{ori}$), two selectable antibiotic resistance genes ($\text{amp}$ and $\text{tet}$), and unique restriction endonuclease cleavage sites.

Recombinant DNA Molecule

A chimeric DNA molecule formed in vitro by enzymatically cleaving a vector and target gene with matching restriction endonucleases and joining their complementary sticky ends via DNA ligase.

Heat Shock Transformation

A method used to induce competent bacterial cells to take up exogenous plasmid DNA from their environment by exposing them to a sudden, transient temperature increase.

Antibiotic Selection

A screening procedure in which transformed bacteria are plated on antibiotic-containing medium so that only cells harboring a plasmid with the corresponding resistance gene can survive and form colonies.

cDNA Library

A library of cloned DNA fragments synthesized from isolated mRNA transcripts using reverse transcriptase, representing exclusively expressed, protein-coding sequences devoid of introns.

Polymerase Chain Reaction (PCR)

An in vitro DNA amplification method that exponentially replicates a target DNA region through repeated automated thermal cycles consisting of denaturation ($94\text{--}95\,^\circ\text{C}$), primer annealing ($\sim 55\text{--}60\,^\circ\text{C}$), and enzymatic extension ($72\,^\circ\text{C}$).

Taq DNA Polymerase

A thermostable enzyme isolated from the thermophilic bacterium Thermus aquaticus that synthesizes new DNA strands in the $5' \rightarrow 3'$ direction, exhibiting an optimal working temperature of $72\,^\circ\text{C}$ and resisting heat denaturation up to $95\,^\circ\text{C}$.

Dideoxynucleotide Triphosphate (ddNTP)

A modified chain-terminating nucleotide lacking hydroxyl ($\text{-OH}$) groups at both the $2'$ and $3'$ positions of the ribose ring, preventing DNA polymerase from forming the subsequent phosphodiester bond during chain elongation.

Sanger Sequencing

A low-throughput gold-standard DNA sequencing technique generating reads of $\sim 500\text{--}1000\text{ bp}$ using DNA polymerase, primers, dNTPs, and a low proportion of fluorescently labeled chain-terminating ddNTPs resolved by capillary gel electrophoresis.

Next-Generation Sequencing (NGS)

A massively parallel sequencing technology that simultaneously sequences millions of short DNA fragments ($50\text{--}300\text{ bp}$) at low cost per base, which are computationally assembled to generate high-depth coverage of whole genomes, exomes, or transcriptomes.

Single-Cell Sequencing

A high-resolution genomics approach wherein individual cells are compartmentalized into barcoded droplets or beads with PCR reagents, enabling single-cell gene expression measurements to detect rare cell types and cellular heterogeneity.

CRISPR-Cas9

A prokaryotic adaptive immune system composed of Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 endonuclease, engineered into a targeted genome-editing tool where Cas9 is guided by complementary RNA to cleave specific double-stranded genomic target sites.

Golden Rice

A genetically modified rice strain engineered to biosynthesize $\beta$-carotene in its endosperm to address dietary vitamin A deficiency, created by introducing a maize phytoene synthase gene (enzyme 1) and a bacterial phytoene desaturase gene (enzyme 2).

Primary Cell Culture

In vitro culture initiated directly from dissociated tissue cells; these cells maintain physiological behaviors closely resembling in vivo biology but possess a finite replicative lifespan of $25\text{--}50$ population doublings before undergoing telomere-dependent replicative senescence.

Immortalized Cell Line

A cultured cell line (often tumor-derived such as HeLa, or virally transformed) capable of dividing indefinitely in vitro through telomerase reactivation and cell-cycle checkpoint inactivation, enabling scalable and high-throughput drug screening.

Organoid

A 3D, self-organizing multicellular in vitro structure grown from stem or progenitor cells that mimics the architecture, multiple differentiated cell types, and physiological function of an intact organ, though lacking vascularization and full organ size.

Hybridoma

An immortalized hybrid cell line produced by fusing an antibody-producing primary B lymphocyte with a cancerous myeloma cell, enabling continuous in vitro generation of identical monoclonal antibodies specific to a single epitope.

Western Blot

An antibody-based molecular technique that identifies whether a target protein is present and measures its molecular weight or post-translational modifications (e.g., phosphorylation) following SDS-PAGE separation and membrane transfer.

ELISA (Enzyme-Linked Immunosorbent Assay)

A quantitative, high-throughput microplate assay using enzyme-conjugated antibodies and chromogenic substrates to measure the precise concentration of a specific dissolved antigen within liquid samples such as plasma, serum, or lysates.

Green Fluorescent Protein (GFP)

A 238-amino-acid reporter protein originally discovered in the jellyfish Aequorea victoria that autocatalytically forms a fluorophore via cyclization and oxidation of a $\text{Ser65-Tyr66-Gly67}$ tripeptide, absorbing light at $488\,\text{nm}$ and emitting green light at $509\,\text{nm}$.

Flow Cytometry

A high-speed biophysical method that uses hydrodynamic focusing to pass single cells through laser beams in single file, measuring forward scatter, side scatter, and multi-color fluorescence on $20{,}000\text{--}50{,}000\text{ cells/second}$.

Immunofluorescence Microscopy

A cell imaging technique that uses fluorophore-conjugated antibodies to visualize the subcellular spatial distribution, morphology, and colocalization of target proteins within intact, fixed cells or tissue sections.

Immunohistochemistry (IHC)

A microscopic staining technique applied to intact histological tissue sections (e.g., FFPE) wherein enzyme-conjugated antibodies (such as HRP or AP) catalyze the deposition of a visible chromogenic precipitate to localize target proteins while preserving tissue architecture.

Tandem Mass Spectrometry (MS/MS)

An unbiased proteomic method where proteins are cleaved into peptides (typically with trypsin), ionized, filtered by precursor mass in MS1, fragmented with inert gas, and analyzed in MS2 by mass-to-charge ($m/z$) ratio to identify sequences, quantify abundances, and localize post-translational modifications.

X-ray Crystallography

A structural biology technique that determines the 3D atomic coordinates ($1\text{--}3\text{ \AA}$ resolution) of macromolecules by analyzing the X-ray diffraction patterns produced by a solid, well-ordered crystal.

Nuclear Magnetic Resonance (NMR) Spectroscopy

A structural biology approach that determines 3D structures ($2\text{--}5\text{ \AA}$ resolution), internal conformational dynamics, and binding kinetics of small macromolecules ($<50\,\text{kDa}$) directly in solution.

Cryo-Electron Microscopy (Cryo-EM)

A structural determination technique where biomolecules are vitrified in liquid ethane into a frozen-hydrated state and imaged with transmission electron microscopy, suited for solving large assemblies ($>150\,\text{kDa}$) and membrane proteins at $2\text{--}4\text{ \AA}$ resolution without crystallization.

AlphaFold

An artificial intelligence computational platform that predicts the 3D atomic coordinates of a protein from its primary amino acid sequence within minutes, serving as a hypothesis generator to guide structural and biochemical experiments.

Cell-Free Extract

A cell-free biological system prepared from unfertilized Xenopus laevis egg cytoplasm lacking nuclei that self-organizes into $300\text{--}400\,\mu\text{m}$ compartments and recapitulates complex cellular behaviors, including DNA replication, mitosis, and cell-cycle oscillations.

Synthetic Lethality

A genetic phenomenon in which the loss or disruption of either of two genes individually allows cell survival, but the simultaneous disruption of both genes leads to cell death (e.g., inhibiting PARP in BRCA-deficient cancer cells).