DNA Extraction, DNA Cloning, and Gene Coding

Comprehensive vocabulary flashcards covering the protocols, vectors, cloning methods, genetic code principles, and mutations presented in the lecture notes on DNA extraction, cloning, and gene coding.

DNA Extraction

The crucial first step in agricultural biotechnology and plant-breeding programs that isolates high-quality genomic DNA from plant tissues to unlock the molecular blueprint needed for downstream applications.

Marker-Assisted Selection (MAS)

A breeding approach where plant DNA is analyzed for genetic markers linked to desirable traits (such as yield, disease resistance, or quality), allowing early-stage seedlings to be screened quickly and efficiently.

Single Nucleotide Polymorphisms (SNPs)

Single-base genetic markers analyzed in plant DNA that are linked to desirable traits and utilized in marker-assisted selection.

Simple Sequence Repeats (SSRs)

Repetitive genetic marker sequences evaluated in plant DNA to facilitate marker-assisted selection for superior traits.

Genome-Wide Association Studies (GWAS)

Analysis tools used alongside linkage maps on diverse plant populations to identify chromosomal regions and genes responsible for complex quantitative traits.

CTAB Method

A DNA extraction protocol using cetyltrimethylammonium bromide (a cationic surfactant) that breaks cell membranes and complexes contaminants; ideal for species rich in polysaccharides or polyphenols.

SDS Method

A simpler and faster DNA extraction protocol using sodium dodecyl sulfate (an anionic detergent) to break open cells and denature proteins; best suited for plants with low levels of interfering metabolites.

Commercial Kit-Based Extraction

A standardized, user-friendly DNA extraction approach that lyses tissue, binds DNA to spin-columns or magnetic beads, and elutes pure DNA within $15-60\,\text{minutes}$.

Organic Solvent Extraction

The phase-separation step in DNA isolation combining lysate with chloroform and isoamyl alcohol ($24:1$), causing DNA to move into the upper aqueous phase while debris, proteins, and lipids settle into the organic layer.

RNase A

An enzyme applied to the aqueous phase during DNA extraction to degrade RNA contaminants, ensuring pure DNA for downstream analyses.

DNA Precipitation (in CTAB Protocol)

The addition of cold isopropanol or ethanol to insolubilize genomic DNA, followed by washing with $70\%$ ethanol to remove residual salts before dissolving the pellet in TE buffer or nuclease-free water.

DNA Purity Ratios (Spectrophotometry)

Absorbance benchmarks where an $A_{260}/A_{280} \approx 1.8$ and an $A_{260}/A_{230} \ge 2.0$ confirm high-purity DNA with minimal protein or organic contamination.

Agarose Gel Electrophoresis

A laboratory technique used to assess the quality of extracted DNA by verifying the presence of intact, high-molecular-weight bands with minimal degradation or smearing.

DNA Cloning

The process of producing multiple identical copies of a specific DNA fragment inside a host cell, also known as gene cloning or molecular cloning.

Cloning Vector

A carrier DNA molecule (usually smaller than $10\,\text{kb}$) capable of autonomous replication in a host cell, containing a cloning site and a selectable marker.

Recombinant DNA (rDNA)

A hybrid DNA molecule formed by the insertion of a target gene of interest into a cloning vector.

DNA Ligase

An enzyme that recognizes and joins the cut ends of DNA fragments by catalyzing the formation of new phosphodiester bonds.

Transformation

The process of introducing recombinant DNA into a host cell so that the cell takes up and expresses the inserted gene of interest.

Competent Cells

Host cells chemically altered (e.g., using cold calcium chloride followed by heat shock) or physically treated to make their membranes permeable to foreign DNA.

Electroporation

A technique that exposes host cells to an electric field to create transient pores in the membrane, facilitating DNA uptake.

Selective Media Plating

Culturing transformed host cells on nutrient agar containing a specific antibiotic so only cells carrying the vector's resistance gene can survive and form colonies.

Plasmids

Small, circular, autonomously replicating prokaryotic DNA molecules used as cloning vectors capable of holding inserts up to approximately $15\,\text{kb}$.

Bacteriophages

Viruses that infect bacteria (such as lambda and M13) utilized as cloning vectors.

Cosmids

Hybrid cloning vectors combining characteristics of both plasmids and bacteriophages, providing higher stability than standard plasmids.

Bacterial Artificial Chromosomes (BACs)

Large-capacity cloning vectors capable of maintaining DNA fragments up to $350\,\text{kb}$ in bacterial cells.

Yeast Artificial Chromosomes (YACs)

Cloning vectors designed for propagating extremely large DNA inserts greater than $1\,\text{Mb}$, commonly used in genome mapping and sequencing projects.

Restriction Enzymes

Bacterial endonucleases that recognize unique palindromic nucleotide sequences and cleave double-stranded DNA at specific recognition sites.

Sticky Ends

Single-stranded overhangs created by staggered restriction enzyme cleavage that can readily anneal to compatible sequences cut by the same enzyme.

Blunt Ends

Non-overhanging double-stranded termini generated by straight cleavage across the DNA duplex by certain restriction enzymes.

TA Cloning

A PCR-based cloning technique where Taq polymerase adds a single adenine ($\text{A}$) overhang to the $3'$ ends of amplified products, allowing direct ligation into vectors with thymidine ($\text{T}$) overhangs.

Ligation-Independent Cloning (LIC)

A cloning method using enzymes with $3'$ to $5'$ exonuclease activity to create cohesive overhangs matching the vector, resulting in constructs with single-stranded nicks repaired in vivo by the host.

Seamless Cloning (SC)

An assembly technique utilizing an enzyme with $5'$ to $3'$ exonuclease activity to generate $3'$ single-stranded overhangs at matching sequences, permitting multiple DNA fragments to be joined into a vector.

Recombinational Cloning

A cloning approach using site-specific DNA recombinases to transfer DNA inserts from an entry clone into a destination vector without restriction digestion and ligation.

Green Fluorescent Protein (GFP)

A fluorescent marker protein originally cloned from jellyfish used extensively to visualize gene expression and protein localization in living cells.

Gene Expression

The multi-step process by which genetic instructions encoded in a gene are transcribed into mRNA and translated into a functional protein product.

Gene Coding

The specific sequence of nucleotide bases within a gene that provides instructions for producing a functional product, typically a protein or functional RNA.

Central Dogma of Molecular Biology

The principle describing the directional flow of genetic information inside biological systems: $\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}$.

Codon

A triplet sequence of three mRNA nucleotides that encodes a specific amino acid or acts as a translational start or stop signal.

Start Codon

The mRNA triplet $\text{AUG}$, which establishes the reading frame, initiates translation, and specifies the amino acid methionine.

Stop Codons

The three non-coding triplet signals ($\text{UAA}$, $\text{UAG}$, and $\text{UGA}$) that cause the ribosome to terminate translation.

Degeneracy (Genetic Code)

The property of the genetic code wherein multiple distinct codons specify the exact same amino acid (e.g., several different codons code for leucine).

Colinearity (Genetic Code)

The principle that the linear order of nucleotides in a coding sequence corresponds directly to the linear arrangement of amino acids in the synthesized polypeptide.

Template Strand

The non-coding DNA strand read in the $3'$ to $5'$ direction by RNA polymerase to synthesize complementary mRNA.

Coding Strand

The DNA strand identical in sequence to the transcribed mRNA (with thymine substituted by uracil in the mRNA), oriented in the $5'$ to $3'$ direction.

Reading Frame

The structural grouping of successive nucleotide triplets into codons during translation, initiated by the start codon.

Silent Mutation

A base substitution that alters a codon but, due to the redundancy of the genetic code, still specifies the identical amino acid (e.g., $\text{GAA} \rightarrow \text{GAG}$, both coding for glutamic acid).

Missense Mutation

A nucleotide alteration that converts a codon into one specifying a completely different amino acid (e.g., $\text{GAA} \rightarrow \text{GUA}$, replacing glutamic acid with valine).

Nonsense Mutation

A single-base substitution that creates a premature stop codon, terminating translation early and generating a truncated, often nonfunctional protein.

Frameshift Mutation

The insertion or deletion of nucleotides in numbers not divisible by three, shifting the reading frame and completely altering all downstream amino acids.