Gene Cloning and Manipulation Review

This set of vocabulary flashcards covers essential terms, enzymes, techniques, and biological systems involved in gene cloning and manipulation as described in the lecture content from Christopher Howe.

Nucleases

Enzymes that break down nucleic acids.

Exonucleolytic activity

The process of dismantling a linear nucleic acid molecule bit by bit from the ends.

Endonucleolytic activity

The process of breaking a nucleic acid molecule into pieces by cutting within the molecule.

Restriction endonucleases

Enzymes that are part of the natural defense mechanisms of bacteria against incoming DNA and recognize particular DNA sequences to cut them.

Hemimethylated DNA

A DNA molecule methylated on only one strand; it is sufficient to confer protection against cleavage by an endonuclease.

Sticky ends

Short single-stranded ends on primarily double-stranded DNA molecules created by a staggered cut from a Type II restriction enzyme.

Blunt ends

Double-stranded DNA ends with no single-stranded overhang, resulting from enzymes that cut both strands at the same place.

Palindromes

DNA sequences that read the same on both strands when read with the same polarity (e.g., 5' to 3').

Isoschizomers

Different restriction enzymes that recognize the same DNA sequence.

Star activity

The relaxation of enzyme specificity, such as $EcoRI$ cutting at $-AATT-$ instead of $-GAATTC-$ in the presence of high glycerol concentrations.

Unit (Enzyme Activity)

The amount of enzyme required to digest $1\,mg$ of a standard type of DNA in $1\,h$ under given conditions.

Genomic DNA

The total DNA prepared from an organism.

Partial digestion

A technique where reaction time or enzyme ratio is adjusted so only a few of the possible restriction sites are cut, raising the average fragment size.

Cos site

A specific $16$-nucleotide site in the bacteriophage lambda genome cut by terminase to leave a $12$-nucleotide single-stranded overhang.

Sonication

A physical method for shearing DNA using high-frequency vibrations.

Phosphatases

Enzymes that hydrolytically remove phosphate groups from DNA molecules, replacing them with hydroxyl groups.

Klenow fragment

A $76\,kDa$ C-terminal fragment of DNA polymerase I containing polymerase and $3'-5'$ exonuclease activities.

Polishing

The process of rendering overhanging DNA ends blunt using polymerase or exonuclease activity.

Thermostable DNA polymerases

Polymerases isolated from extrêmement thermophilic bacteria like $Thermus\,aquaticus$ ($Taq$) used in PCR.

Reverse transcriptases (RTases)

RNA-dependent DNA polymerases that synthesize DNA from an RNA template.

Terminal transferase

A template-independent polymerase from calf thymus that attaches deoxyribonucleotides one by one to the $3'$ end of a DNA molecule.

Exonuclease III (exoIII)

An enzyme with $3'-5'$ exonuclease activity on double-stranded DNA that will not attack protruding $3'$ ends.

$S1$ nuclease

A single-strand-specific exonuclease from $Aspergillus\,oryzae$.

Ligation

The alignment of the ends of two DNA molecules and the formation of a covalent phosphodiester bond between them.

Nick

A break in the sugar-phosphate backbone of a double-stranded DNA molecule that can be sealed by a phosphodiester bond.

Gap

A region in a DNA strand where nucleotides are missing, which cannot be sealed by ligation alone.

Topoisomerase

An enzyme that alters the degree of supercoiling and can also function as a fast DNA ligase.

Transformation

The introduction of DNA into a recipient host organism.

Competence

The natural or induced ability of bacteria to take up exogenous DNA molecules.

Conjugation

A process where plasmids are passed from one cell to another through direct physical contact via a sex pilus.

Transfection

A term often referring to the uptake of viral DNA or the transformation of eukaryotic cells.

Electroporation

Inducing cells to take up DNA by subjecting them to an electric shock that briefly makes the membrane permeable.

Biolistic transformation

A physical transformation method using a particle gun to fire DNA-adsorbed microprojectiles into recipient cells.

Ethidium bromide

A flat, hydrophobic molecule that intercalates between DNA bases and fluoresces brightly under UV light.

Southern blot

A technique to transfer DNA from an electrophoresis gel to a membrane for hybridization with a probe.

Northern blot

A technique to transfer RNA from an electrophoresis gel to a membrane for analysis.

Microarrays

Ordered collections of large numbers of different DNA sequences immobilized onto a solid-phase support, often used for transcriptomic analysis.

Polymerase Chain Reaction (PCR)

A direct enzymatic process for amplifying specific DNA sequences using primers and a heat-stable DNA polymerase.

Primer dimers

Artefacts formed in PCR when two primers anneal to each other and are extended by DNA polymerase.

Jumping PCR

A phenomenon where a truncated amplification product anneals to a different DNA fragment in a subsequent round, allowing synthesis of a full product from degraded DNA.

Hot-start PCR

A method to prevent non-specific primer annealing by keeping a reaction component unavailable until the first cycle reaches its melting temperature.

Nested PCR

A two-stage PCR process where the second set of primers anneals within the product of the first PCR to improve specificity.

Inverse PCR (IPCR)

A method used to amplify DNA sequences outside the region of known primer sites by circularizing the template.

Real-time PCR (qPCR)

A technique to quantify DNA accumulation during the PCR process using fluorescent dyes or probes.

Asymmetric PCR

A PCR variation using unequal primer concentrations to preferentially amplify one strand, resulting in single-stranded DNA.

Vector

A DNA molecule, such as a plasmid, used for propagating foreign DNA in a host cell.

Alpha-complementation

Intragenic complementation between the $lacZ'$ minigene product on a plasmid and a deleted $lacZ$ gene in the host to produce functional beta-galactosidase.

Shotgun cloning

The generation of recombinants by randomly cloning fragments of the total DNA of an organism.

Genomic library

A random collection of host colonies containing recombinant DNA molecules representing an organism's entire genome.

Selectable marker

A gene, like ampicillin resistance ($ampR$), used to distinguish cells that have taken up a vector from those that have not.

Bacterial artificial chromosome (BAC)

A vector based on the F factor of $E.\,coli$ used for cloning very large DNA inserts ($100-300\,kbp$).

Replicative form (RF)

The double-stranded circular DNA molecule formed during the life cycle of bacteriophage M13.

Phage display

A system where foreign peptides are fused to phage coat proteins and expressed ("displayed") on the outside of the virus particle.

Phagemid

A plasmid carrying a filamentous phage replication origin, allowing it to be packaged as single-stranded DNA if a helper phage is present.

Plaque

A hole in a bacterial lawn resulting from infected cells that have lysed or had their growth retarded by phage.

Cosmid

A plasmid replacement vector containing the lambda $cos$ site, allowing it to be packaged into phage coats for efficient delivery.

Fosmid

A cosmid vector that uses an origin of replication from the F plasmid to maintain a low copy number.

cDNA library

A collection of clones made from DNA copies of mRNA sequences, representing expressed genes.

Normalization (Library)

A technique to increase the representation of rare mRNAs in a library by exploiting hybridization kinetics to remove abundant sequences.

Chromosome walking

Successive rounds of genomic library screening to assemble an ordered collection of overlapping clones along a chromosome.

Transposon tagging

Using a transposable element as a hybridization probe to identify and clone a gene into which it has inserted and caused a mutation.

Positional cloning

The process of obtaining a clone for a gene based on its location on a genetic map.

Reporter gene

A gene whose expression level can be easily assessed (e.g., $GFP$, luciferase) to report on the activity of a promoter or regulatory sequence.

Two-hybrid screening

A technique used to identify protein-protein interactions by linking a DNA-binding domain and an activation domain via two interacting proteins.

Reverse genetics

An experimental approach that starts with a cloned gene and inactivates it to determine its biological function.

Protein engineering

Directly altering specific parts of a protein to probe the relationship between its structure and function.

Linker scanning

A technique where regions of a sequence are deleted and replaced with a DNA fragment of equal length to maintain spacing between control elements.

Auxotrophy

The nutritional requirement of a mutant for a specific metabolite that it can no longer synthesize.

Gene disruption

Inactivating a chromosomal gene by replacing it with a non-functional copy through homologous recombination.

RNA interference (RNAi)

A cellular mechanism where small double-stranded RNAs lead to the breakdown of corresponding endogenous single-stranded mRNA.

Short interfering RNAs (siRNAs)

Short fragments of approximately $22$ nucleotides generated by Dicer as part of the RNAi pathway.

Ribozyme

An RNA molecule with direct catalytic activity, such as self-cleavage.

Fusion protein

A hybrid protein encoded by a vector where the N-terminal is provided by the vector and the rest is encoded by an inserted sequence.

Tag

A short peptide region (e.g., His-tag, FLAG-tag) fused to a protein of interest to facilitate detection or purification.

Inclusion bodies

Insoluble aggregates of incorrectly folded proteins often found when expressing foreign genes at high levels in $E.\,coli$.

Inteins

Peptides that carry out post-translational self-splicing, which can be modified for use in protein purification.

Gene trapping

A method using a promoterless reporter gene to identify and clone sequences that are specifically expressed in a particular tissue.

Shuttle vector

An artificially constructed plasmid containing replication origins for two different species, such as $E.\,coli$ and yeast.

Yeast artificial chromosome (YAC)

A linear vector containing a centromere ($CEN$), telomeres ($TEL$), and an origin of replication ($ARS$) used to clone megabase-sized DNA in yeast.

T-DNA

The DNA region of the Ti plasmid from $Agrobacterium$ that is transferred into the plant cell nucleus and integrated into its genome.

Knock-out mice

Mice in which a specific gene has been inactivated through gene targeting in embryonic stem cells.