BISC 300 chapter 15 textbook

Restriction Endonucleases (Restriction Enzymes)

Bacterial defense enzymes that recognize specific DNA sequences and cleave foreign viral DNA to halt viral replication.

Palindromic Sequence (DNA)

A double-stranded DNA sequence that reads the same 5′ to 3′ on the leading strand as it does 5′ to 3′ on the complementary strand (e.g., 5′-GAATTC-3′ / 3′-CTTAAG-5′).

DNA Methylation

The bacterial mechanism of adding methyl groups to genomic DNA at recognition sites to protect it from being degraded by its own restriction enzymes.

Recombinant DNA

DNA created artificially by combining genetic material from multiple sources using restriction digest and ligation.

EcoRI

A specific restriction enzyme isolated from E. coli that cleaves at the palindromic sequence 5′-GAATTC-3′.

Plasmids

Small, circular pieces of extrachromosomal bacterial DNA used in biotechnology as vectors to carry a gene of interest into target cells.

Origin of Replication (Ori)

A specific plasmid sequence that allows the host cell's machinery to duplicate the plasmid.

Promoter

A sequence preceding the target gene that controls transcription; can be constitutive (always on, like CMV) or inducible (controlled by chemical signals).

Inducible Promoter

A promoter system turned on or off by a specific compound, such as the Tet-On system (activated by tetracycline) or Tet-Off system (inhibited by tetracycline).

Selection Marker / Reporter

A plasmid gene (such as antibiotic resistance or a color marker) that allows researchers to identify cells that successfully took up the plasmid.

Polymerase Chain Reaction (PCR)

An in vitro technique developed by Kary Mullis that uses temperature cycling to exponentially replicate a specific region of DNA.

Taq Polymerase

A heat-stable DNA polymerase isolated from Thermus aquaticus that survives the high-temperature denaturation steps in PCR.

dNTPs (Deoxynucleoside Triphosphates)

The individual nucleotide building blocks (A, T, G, C) used by polymerase to build new DNA strands during PCR.

Quantitative PCR (qPCR / Real-time PCR)

A PCR variation using fluorescent dyes or probes to measure DNA amplification in real time for comparative quantification of starting material.

Multiplex PCR

A PCR protocol that amplifies multiple distinct target DNA sequences at the same time in a single reaction tube.

Reverse Transcriptase

An enzyme derived from retroviruses that synthesizes complementary DNA (cDNA) from an RNA template.

Complementary DNA (cDNA)

DNA synthesized from processed mRNA using reverse transcriptase; lacks non-coding introns, allowing eukaryotic genes to be expressed in prokaryotes.

RT-PCR (Reverse Transcription PCR)

A process combining reverse transcription and PCR to convert mRNA into cDNA and then amplify it to quantify gene expression levels.

CRISPR Cas-9

A bacterial adaptive immune system adapted for targeted genome editing using a guide RNA to direct the Cas-9 endonuclease to cut specific DNA sequences.

Guide RNA (gRNA)

An engineered RNA strand in the CRISPR Cas-9 system that matches a Target genomic sequence to direct the Cas-9 enzyme to the exact edit location.

Bioproduction

The use of living microorganisms or cultured cells to produce organic products, pharmaceuticals, or industrial proteins.

Upstream Processing

The first stage of bioproduction involving gene identification, RT-PCR, cDNA creation, plasmid construction, and host transformation.

Protein Production Stage

The middle stage of bioproduction where engineered microbes are grown in large vessels called bioreactors under controlled continuous conditions.

Bioreactor

A specialized culture vessel equipped with automated systems to regulate temperature, aeration, agitation, nutrients, and waste outflow.

Downstream Processing

The final stage of bioproduction involving target protein extraction, column chromatography purification, quality assurance testing, and packaging.

Daniel Nathans

Nobel laureate (1978) recognized alongside Werner Arber and Hamilton Smith for the discovery and application of restriction endonucleases.

Kary Mullis

Nobel laureate (1993 in Chemistry) recognized for inventing the Polymerase Chain Reaction (PCR).

Emmanuelle Charpentier & Jennifer Doudna

Nobel laureates (2020 in Chemistry) recognized for adapting the bacterial CRISPR Cas-9 system into a tool for gene editing.