Recombinant DNA Technology and Molecular Tools
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A comprehensive vocabulary review containing 100 practice flashcards on recombinant DNA technology, cloning vectors, restriction endonucleases, molecular techniques, and ethical applications.
111 Terms
Recombinant DNA Technology
The collection of techniques used to perform genetic engineering, including isolating genes, inserting them into vectors, and expressing them in host organisms.
Primary Goals of Recombinant DNA Technology
To identify and isolate a specific gene of interest, insert the gene into a vector to create a recombinant molecule, and produce large quantities of the gene and its product.
Genetic Engineering
The direct manipulation of an organism's genes using recombinant DNA technology to alter its traits or produce specific biological products.
Restriction Enzymes (Restriction Endonucleases)
Enzymes produced by bacterial cells that cleave double-stranded DNA at specific recognition base sequences.
Natural Biological Function of Restriction Enzymes
To protect bacteria against viral invasion by cleaving and destroying foreign viral DNA upon entry.
Restriction Site
A specific sequence of nucleotide bases recognized and cleaved by a restriction endonuclease.
Palindromic Recognition Sequence
A sequence of double-stranded DNA that reads identically in the 5′→3′ direction on both complementary strands.
Sticky Ends
Staggered, single-stranded overhangs produced by restriction enzyme cleavage that readily form complementary hydrogen bonds with matching cohesive sequences.
Blunt Ends
Fully base-paired DNA ends produced by straight cuts across both DNA strands at the cleavage site, lacking single-stranded overhangs.
Type II Restriction Endonucleases
A class of restriction enzymes that recognize specific symmetrical sequences (commonly 4, 6, or 8 base pairs long) and cleave predictably within or immediately adjacent to that site.
Four-Base Recognition Sequence
A restriction recognition site composed of four nucleotides, such as 5′-AGCT-3′ recognized by AluI.
Six-Base Recognition Sequence
A restriction site composed of six nucleotides, such as 5′-GGATCC-3′ (BamHI) or 5′-GAATTC-3′ (EcoRI).
Eight-Base Recognition Sequence
A restriction recognition sequence composed of eight nucleotides, such as 5′-GCGGCCGC-3′ recognized by NotI.
AluI
A Type II restriction enzyme derived from Arthrobacter luteus that produces blunt ends upon cleavage.
Arthrobacter luteus
The bacterial microbial source from which the restriction endonuclease AluI is isolated.
AluI Cleavage Pattern
Cleaves the symmetrical sequence 5′-AGCT-3′ / 3′-TCGA-5′ between G and C to generate blunt ends: 5′-AG CT-3′ and 3′-TC GA-5′.
BamHI
A Type II restriction endonuclease isolated from Bacillus amyloliquefaciens H that generates cohesive sticky ends with a 5′ overhang.
Bacillus amyloliquefaciens H
The microbial source of the restriction endonuclease BamHI.
BamHI Cleavage Pattern
Cleaves the sequence 5′-GGATCC-3′ / 3′-CCTAGG-5′ between the two adjacent guanines (G and G), producing 5′-G GATCC-3′ and 3′-CCTAG G-5′.
EcoRI
A Type II restriction endonuclease isolated from Escherichia coli that cleaves DNA to generate sticky ends with four-base 5′ overhangs.
Escherichia coli (Restriction Enzyme Source)
The bacterium that serves as the natural microbial source of the restriction enzyme EcoRI.
EcoRI Cleavage Pattern
Recognizes the sequence 5′-GAATTC-3′ / 3′-CTTAAG-5′ and makes staggered cuts between G and A on each strand.
EcoRI Cleavage Products
The fragments resulting from EcoRI digestion, displaying single-stranded 5′-AATT-3′ sticky overhangs: 5′-G AATTC-3′ and 3′-CTTAA G-5′.
HindIII
A Type II restriction enzyme derived from Haemophilus influenzae d that cleaves DNA leaving staggered sticky ends.
Haemophilus influenzae d
The bacterial species and strain serving as the natural source of the restriction endonuclease HindIII.
HindIII Cleavage Pattern
Cleaves the sequence 5′-AAGCTT-3′ / 3′-TTCGAA-5′ between adjacent adenines (A and A), yielding 5′-A AGCTT-3′ and 3′-TTCGA A-5′.
NotI
An 8-base cutter Type II restriction enzyme derived from Nocardia otitidis-caviarum that cleaves rare GC-rich recognition sequences.
Nocardia otitidis-caviarum
The microbial bacterial source of the rare-cutting restriction enzyme NotI.
NotI Cleavage Pattern
Cleaves the 8-base palindromic sequence 5′-GCGGCCGC-3′ / 3′-CGCCGGCG-5′ between C and G to generate 5′-GC GGCCGC-3′ and 3′-CGCCGG CG-5′.
SalI
A Type II restriction enzyme isolated from Streptomyces albus that cuts a 6-base recognition sequence producing cohesive sticky ends.
Streptomyces albus
The bacterial microbial source of the restriction endonuclease SalI.
SalI Cleavage Pattern
Cleaves the sequence 5′-GTCGAC-3′ / 3′-CAGCTG-5′ between G and T, producing 5′-G TCGAC-3′ and 3′-CAGCT G-5′.
DNA Ligase
An enzyme that seals nicks in the sugar-phosphate backbone by catalyzing phosphodiester bond formation between complementary annealed DNA fragments.
Recombinant DNA Molecule
A DNA molecule engineered in vitro by joining DNA fragments from two different sources or organisms into a single cloning vector.
Cloning Vector
A self-replicating DNA molecule (such as a plasmid or virus) used as a vehicle to transfer foreign genetic material into a host cell for cloning or expression.
Gene Cloning: Step 1
Isolation of the source genomic or target DNA containing the specific gene to be cloned.
Gene Cloning: Step 2
Generation of target DNA fragments using restriction endonucleases or polymerase chain reaction (PCR).
Gene Cloning: Step 3
Generation of a recombinant molecule by inserting the isolated DNA fragments into an open, linearized cloning vector.
Gene Cloning: Step 4
Introduction of the recombinant vector molecule into a competent recipient host cell via transformation or electroporation.
Gene Cloning: Step 5
Selection and screening of transformed host cells containing the recombinant gene of interest using antibiotic selection or blue/white screening.
Gene Cloning: Step 6
Expression of the cloned gene within the host to produce and isolate a purified recombinant protein.
Vector Capacity
A required vector quality referring to its physical ability to carry and maintain a significant insert piece of donor DNA.
Vector Host Acceptance
A necessary property of a cloning vector that allows it to be readily taken up, maintained, and replicated by the chosen host organism.
Selectable Marker
A specific gene on a cloning vector (such as an antibiotic resistance gene) that confers a phenotype enabling the identification and isolation of transformed host cells.
Rapid Growth Rate (Cloning Host Feature)
A desirable property of cloning host strains that enables quick cell division and short generation times.
Ordinary Culture Cultivation (Cloning Host Feature)
The desirable capability of a cloning host to grow robustly in large volumes using standard, inexpensive laboratory growth media.
Nonpathogenic Cloning Host
A safety requirement ensuring that recipient laboratory host cells (such as standard E. coli) do not cause disease in humans or animals.
Mapped Host Genome
A desirable host trait where the entire genomic sequence and background genetics of the host cell are fully known and documented.
Multigenerational Foreign Gene Stability
The capacity of a cloning host to faithfully maintain, replicate, and transmit recombinant plasmid or phage vectors across numerous cell divisions without loss.
Recombinant DNA Cloning Vectors
A spectrum of molecular vehicles—including plasmids, bacteriophages, cosmids, BACs, and YACs—designed with specific insert capacities and replication machinery.
Plasmid Vector
A small, circular extrachromosomal DNA molecule that replicates independently of the host chromosome, maintaining high copy numbers in a single cell.
Plasmid Insert Size Limit
The functional insert capacity of plasmid cloning vectors, typically limited to DNA fragments smaller than 20kb.
pBR322
A classic standard plasmid cloning vector designed for E. coli that maintains independent replication and carries antibiotic resistance genes.
pUC19
A high-copy-number plasmid cloning vector containing an ampicillin resistance marker and a multiple cloning site located inside the lacZ gene.
Bacteriophage Vector
A recombinant cloning vehicle based on viral genomes that can accommodate inserts ranging from 9kb to 25kb.
Lambda Phage Vectors (\lambda 1059, \lambda gt11, EMBL3)
Modified bacteriophage lambda vectors packaged into viral particles to efficiently infect bacterial hosts with 9–25kb DNA inserts.
M13mp18
A modified single-stranded DNA bacteriophage vector engineered to generate either double- or single-stranded DNA in host bacterial cells.
Cosmid Vector
A hybrid plasmid cloning vector incorporating lambda cos sites, capable of holding 30–47kb inserts, packaged into phage coats, and replicated as a plasmid.
pJC720 and pSupercos
Specific commercial examples of cosmid vectors designed for packaging large genomic fragments (30–47kb) into lambda capsids.
Bacterial Artificial Chromosomes (BACs)
Cloning vectors derived from the E. coli fertility (F) plasmid that stably carry very large foreign DNA inserts between 75kb and 300kb.
pBAC108L
A representative bacterial artificial chromosome (BAC) vector based on a modified F plasmid used for cloning DNA fragments up to 300kb.
Yeast Artificial Chromosomes (YACs)
Artificial chromosome vectors that replicate in Saccharomyces cerevisiae and accommodate the largest DNA inserts, ranging from 100kb to 1,000kb.
pYAC
A model yeast artificial chromosome cloning vector used to replicate massive linear eukaryotic DNA inserts in yeast.
Saccharomyces cerevisiae (Host)
The eukaryotic unicellular budding yeast host species utilized to propagate and maintain yeast artificial chromosomes (YACs).
Ampicillin Resistance Gene (AmpR)
A selectable marker that encodes beta-lactamase, conferring host resistance to ampicillin and allowing only successfully transformed bacteria to grow.
lacZ Gene
A gene on cloning plasmids encoding beta-galactosidase whose coding region contains restriction enzyme cleavage sites used for insertional inactivation.
Insertional Inactivation
The disruption of a functional marker gene's open reading frame (such as lacZ) caused by the successful ligation and insertion of foreign DNA.
Blue-White Screening
A visual selection method where bacteria containing non-recombinant vectors produce functional beta-galactosidase and turn blue, while recombinants with interrupted lacZ form white colonies.
Blue Colonies (in lacZ Screening)
Bacterial colonies carrying recircularized vectors without foreign DNA inserts; the intact lacZ gene produces active beta-galactosidase that metabolizes substrate into a blue color.
White Colonies (in lacZ Screening)
Bacterial transformants carrying recombinant plasmids with cloned DNA inserts interrupting lacZ, preventing enzyme synthesis.
Heterologous Gene
A gene originating from one species that is cloned into and expressed within a completely different host organism.
Expression Vector
A specialized cloning vector engineered with strong host-recognized promoters and regulatory signals that drive high-level transcription and translation of cloned genes.
Promoter Incompatibility Barrier
The inability of bacterial RNA polymerase to transcribe eukaryotic genes unless an upstream bacterial promoter recognized by the host is provided.
Ribosome Binding Barrier
The translational requirement in bacterial expression for an appropriate prokaryotic leader sequence (Shine-Dalgarno sequence) upstream of the start codon.
Intron Removal Requirement
The mandatory removal of eukaryotic non-coding intervening sequences (introns) before expression in bacteria, because prokaryotes lack RNA splicing machinery.
Polyhistidine Tag (His-tag)
A recombinant affinity peptide tag consisting of six histidine residues (cat cat cac cat cac cac) added to either the N- or C-terminus of a target protein.
N-Terminal His-Tag Sequence
A His-tag placed immediately downstream of the ATG start codon (5′-ATG cat cat cac cat cac cac NNNN...-3′) at the amino-terminus.
C-Terminal His-Tag Sequence
A His-tag inserted immediately prior to the TAG stop codon (5′-...NNNN cat cat cac cat cac cac TAG-3′) at the carboxyl-terminus.
Immobilized Metal Affinity Chromatography (IMAC)
A protein purification technique utilizing a column packed with nickel- or cobalt-coated resin that selectively binds the polyhistidine tag on recombinant proteins.
Column Wash Buffer (His-tag Purification)
A buffer applied to an IMAC column to remove unwanted, unbound host proteins and cellular debris while leaving the His-tagged protein attached.
Imidazole
A compound structurally similar to histidine used in solution to competitively displace and release His-tagged proteins from nickel- or cobalt-coated resin columns.
Polymerase Chain Reaction (PCR)
An in vitro molecular technique used to rapidly amplify specific target DNA sequences through repeated cycles of denaturation, annealing, and enzymatic extension.
Taq Polymerase
A thermostable DNA polymerase isolated from Thermus aquaticus that synthesizes new complementary DNA strands during the extension step of PCR.
dNTPs (Deoxynucleoside Triphosphates)
The four fundamental nucleoside triphosphate building blocks (dATP, dCTP, dGTP, dTTP) required by DNA polymerase to synthesize new DNA strands.
PCR Primers
Two short single-stranded oligonucleotides present in high concentration that flank and bind specifically to complementary sequences at the ends of the target DNA region.
Denaturation Step (PCR)
The initial phase of a PCR cycle where reaction temperature is elevated briefly to break hydrogen bonds and separate double-stranded template DNA into single strands.
Annealing Step (PCR)
The PCR phase where the reaction is cooled to permit forward and reverse primers to base-pair specifically with the complementary flanking borders of target single-stranded DNA.
Extension Step (PCR)
The phase of PCR where DNA polymerase synthesizes new complementary DNA by adding nucleotides to the 3′ end of each annealed primer.
Amplification Yield in Early PCR Cycles
Cycle 1 produces 2 molecules, Cycle 2 produces 4 molecules, and Cycle 3 produces 8 molecules (with exactly 2 matching the precise target sequence boundaries).
Agarose Gel Electrophoresis
A laboratory method that separates charged nucleic acid molecules across an agarose matrix in an electric field according to molecular size.
Positive Electrode (Anode) Migration
The directional movement of negatively charged DNA molecules through an agarose gel toward the positive pole when an electric current is applied.
DNA Size Markers (DNA Ladder)
A standard set of pre-determined DNA fragments of known lengths (e.g., 5,160bp down to 750bp) loaded to estimate the sizes of unknown experimental samples.
DNA Smear
A continuous, diffuse trail of staining observed on an agarose gel when complex genomic DNA containing countless cleavage sites is digested by restriction enzymes.
Southern Blotting
A hybridization method combining gel electrophoresis, capillary membrane transfer, and labeled probe binding to detect specific DNA sequences.
Capillary Blotting Transfer Assembly
An experimental arrangement using a buffer reservoir, sponge wick, agarose gel, DNA-binding membrane, paper towels, and a weight to draw buffer upward and transfer DNA.
Radioactive Probe (Southern Blotting)
A labeled single-stranded nucleic acid sequence incubated with a membrane to identify complementary target bands via hybridization and autoradiography.
Genomic Library
A collection of clone-containing microbial cells that collectively harbor fragments representing the entire genome of an organism inserted into cloning vectors.
CRISPR-Cas9 System
A prokaryotic adaptive immune defense mechanism against bacteriophage infection adapted as a tool to edit, inactivate, or replace target genes in various organisms.
CRISPR Spacers
Short fragments of captured foreign viral or bacteriophage DNA inserted between chromosomal repeats in bacteria to preserve a molecular memory of past infections.
crRNA (CRISPR RNA)
Small RNA transcripts derived from transcribed CRISPR repeat-spacer units that pair with Cas endonucleases to guide targeted cleavage of matching foreign DNA.