Chapter 18: Recombinant DNA Technology and Molecular Biotechnology

Comprehensive vocabulary flashcards covering the core concepts, laboratory techniques, enzymes, vectors, sequencing technologies, and genetic engineering tools in Chapter 18.

Gene Cloning

The process of producing many identical copies of a specific gene or DNA segment of interest.

Restriction Endonucleases (Restriction Enzymes)

Enzymes that naturally occur in bacteria as a defense mechanism against viral/phage DNA and cleave double-stranded DNA at specific internal nucleotide sequences.

Restriction Site

A specific, usually palindromic nucleotide sequence recognized and cut by a restriction enzyme.

Sticky Ends (Cohesive Ends)

Short, single-stranded overhangs created by staggered cuts of restriction enzymes that can base-pair with complementary ends.

Blunt Ends

Clean, straight cuts across both DNA strands made by restriction enzymes that leave no single-stranded overhangs.

DNA Ligase

An enzyme used in recombinant DNA technology that catalyzes the formation of phosphodiester bonds to permanently seal DNA fragments together.

Cloning Vector

A DNA molecule (such as a plasmid or viral vector) used as a vehicle to artificially carry foreign genetic material into another cell.

Origin of Replication (ori)

A critical vector sequence that allows the host cell to replicate the plasmid independently of its own chromosome.

Selectable Marker Gene

A gene (such as antibiotic resistance) on a cloning vector that allows researchers to select for cells containing the vector.

Insertional Inactivation

The disruption of a gene's function when foreign DNA is inserted directly into it.

Blue Colony (Blue-White Screening)

A colony containing an intact lacZ gene capable of producing beta-galactosidase, indicating a non-recombinant plasmid that turns blue in the presence of X-gal.

White Colony (Blue-White Screening)

A colony containing a disrupted lacZ gene, indicating a successful recombinant plasmid containing inserted foreign DNA.

Genomic Library

A collection of recombinant clones that collectively contain the entire genomic DNA of an organism.

cDNA Library

A collection of clones containing only complementary DNA copies of mRNA molecules expressed in a specific cell type.

Reverse Transcriptase

The enzyme essential for synthesizing cDNA molecules from an mRNA template.

Bacterial Intron Splicing Deficiency

The reason eukaryotic genomic genes often fail to function in bacteria, as bacteria lack spliceosomes and cannot remove non-coding introns.

Polymerase Chain Reaction (PCR)

A laboratory technique used to amplify specific segments of DNA exponentially using a template, primers, dNTPs, Taq polymerase, and a magnesium buffer.

Denaturation Step (PCR)

The thermal step at $94\text{--}98^\circ\text{C}$ where high heat breaks hydrogen bonds, separating double-stranded DNA into single strands.

Annealing Step (PCR)

The thermal step at $50\text{--}65^\circ\text{C}$ during which primers bind to complementary sequences on the single-stranded DNA template.

Extension Step (PCR)

The thermal step at $72^\circ\text{C}$ during which Taq polymerase synthesizes a new DNA strand complementary to the template.

Taq Polymerase

A heat-stable (thermophilic) DNA polymerase that does not denature at the high temperatures used during PCR denaturation.

Gel Electrophoresis

A method that separates DNA fragments by size/length as they migrate through an agarose gel toward the positive electrode due to their negatively charged phosphate backbone.

Sanger (Dideoxy) Sequencing

A sequencing method that uses dideoxynucleotides to randomly terminate DNA synthesis, generating fragments of varying lengths separated by capillary electrophoresis.

Dideoxynucleotide (ddNTP)

A modified nucleotide lacking the critical $3'\text{-OH}$ group on the sugar ring, which prevents DNA polymerase from adding subsequent nucleotides and stops chain elongation.

Next-Generation Sequencing (NGS)

A high-throughput sequencing method that sequences millions of DNA molecules simultaneously in parallel.

Southern Blotting

A molecular technique used to detect specific DNA sequences within a DNA sample using a labeled probe.

Northern Blotting

A molecular technique used to detect specific RNA molecules to analyze gene expression levels.

Western Blotting

A molecular technique used to detect specific proteins within a protein sample using antibodies.

Site-Directed Mutagenesis

The introduction of specific, targeted nucleotide substitutions or deletions into a cloned gene to study protein function.

CRISPR-Cas9 (Bacterial Immune System)

An adaptive immune system in bacteria and archaea that stores viral DNA spacers to target and destroy future viral infections.

Single Guide RNA (sgRNA)

An engineered RNA component of CRISPR-Cas9 whose spacer sequence is complementary to target DNA, guiding the Cas9 enzyme to the precise cleavage site.

Cas9 Endonuclease

A protein component of CRISPR-Cas9 that acts as molecular scissors to create a double-strand break at the targeted genomic site.

PAM (Protospacer Adjacent Motif) Sequence

A short downstream DNA sequence required by Cas9 to recognize and bind the target site.

Non-Homologous End Joining (NHEJ)

An error-prone DNA repair pathway that glues broken ends together, frequently causing indels and gene knockouts.

Homology-Directed Repair (HDR)

A precise DNA repair pathway that uses a homologous donor template to repair a double-strand break accurately.

Genetically Modified Organism (GMO)

Any organism whose genetic material has been altered using genetic engineering techniques.

Transgenic Organism

A genetically modified organism that specifically contains inserted genetic material originating from a different species.

Molecular Pharming

The use of transgenic plants or animals to manufacture pharmaceutical proteins.

Gene Therapy

The introduction of normal or modified genes into a patient's cells to treat a genetic disorder.

Ex Vivo Gene Therapy

A therapeutic approach that modifies cells outside the patient's body before reintroducing them.

In Vivo Gene Therapy

A therapeutic approach that delivers the corrective gene directly inside the patient's body.

DNA Microarray

A tool used to measure the relative expression levels of thousands of genes simultaneously using fluorescently labeled cDNA.

RNA-Seq

A sequencing-based technique that maps and quantifies all RNA transcripts in a sample directly, offering a wider dynamic range and novel transcript detection without needing a pre-designed reference genome.

Metagenomics (Environmental Genomics)

The sequencing and analysis of total DNA collected directly from complex environmental samples.

Short Tandem Repeats (STRs)

Short, highly polymorphic repeating sequences of non-coding DNA found throughout the genome, amplified via PCR and sized for forensic identification.

Gene Knockout

The complete inactivation of a specific gene to study the resulting phenotypic consequences.

RNA Interference (RNAi)

A biological pathway where small non-coding RNAs bind to target mRNAs to promote their degradation or block their translation.

Proteomics

The systematic study of all the proteins expressed by a genome.