Recombinant DNA Technology and Its Applications

Comprehensive vocabulary flashcards covering the definitions, enzymes, techniques, and applications of recombinant DNA technology based on the lecture notes.

Recombinant DNA Technology

The isolation and manipulation of DNA to produce chimeric (hybrid) molecules.

Gene cloning

The process of producing multiple identical copies of a DNA sequence.

Genetic engineering

The deliberate modification of an organism's genetic material.

Gene splicing

The cutting and joining of DNA fragments from different sources.

Restriction Enzymes

Special types of enzymes that cut DNA from any source at specific locations, usually recognizing palindromic sequences of $4-8$ bps.

Sticky ends

DNA fragments with cohesive ends resulting from restriction endonuclease digestion; they may reconnect or anneal with tandem heterogeneous inserts.

Blunt ends

DNA fragments resulting from digestion where no overhanging bases exist; they offer no control over orientation and no easy way to retrieve the insert.

EcoRI

A restriction endonuclease from Escherichia coli RY13 that recognizes the sequence $GAATTC$ and produces sticky ends.

Terminal transferase

An enzyme used to add homopolymer tails, such as poly d(G) or poly d(C), to the $3'$ ends of DNA molecules to facilitate annealing.

Restriction Enzyme Mapping

A technique used to deduce the sites of restriction enzyme cleavage by digesting a gene with several enzymes and separating fragments by gel electrophoresis.

Gel Electrophoresis

A method to separate DNA fragments by size, where DNA migrates from the Cathode (-) to the Anode (+).

Clone

A large population of identical molecules, bacteria, or cells that arise from a common ancestor.

Plasmids

Small, circular, duplex DNA molecules that replicate independently from bacterial DNA and often confer antibiotic resistance.

Bacteriophages

Viruses that infect bacteria and have linear DNA molecules; they can accept inserts of $10-20$ kb.

Cosmids

Plasmids containing cos sites required for packaging lambda DNA; they can carry inserts ranging from $35$ to $50$ kb.

Expression Vector

A vector designed for the synthesis of the protein coded by the introduced gene, containing active inducible promoters and translation initiation codons.

DNA Ligase

An enzyme that joins two DNA fragments by forming phosphodiester bonds; T4 DNA ligase can join both sticky and blunt ends.

Reverse Transcriptase

An enzyme used to synthesize cDNA from an mRNA template; essential for cloning eukaryotic genes.

Alkaline Phosphatase (SAP/CIP)

An enzyme that removes $5'$ phosphates from a vector to prevent self-ligation.

Bacterial Transformation

The process of introducing foreign DNA into a bacterial cell, often achieved via heat shock or electroporation.

pBR322

A plasmid used in recombinant screening; inserting DNA into its PstI site disrupts ampicillin resistance.

Genomic library

A collection of cloned DNA fragments prepared from the total DNA of a cell line or tissue.

cDNA library

A library consisting of complementary DNA copies of the population of mRNAs in a tissue.

Probes

Short, single-stranded pieces of DNA or RNA labeled with radioisotopes like $^{32}P$ or fluorescent dyes to detect complementary sequences.

Southern blotting

A method invented by Edwin Mellor Southern to detect a specific DNA sequence in a sample using electrophoresis and membrane hybridization.

Northern blotting

A blot transfer procedure used for the detection and analysis of RNA.

Western blotting

A blot transfer procedure used to detect specific proteins using antibodies.

PCR (Polymerase Chain Reaction)

A rapid method of amplifying a target DNA sequence using oligonucleotide primers and a thermostable DNA polymerase.

Denaturation (PCR)

Step 1 of PCR where heat ($94-98^\circ\text{C}$) breaks hydrogen bonds to separate double-stranded DNA into single templates.

Primer annealing (PCR)

Step 2 of PCR where temperature drops to $50-65^\circ\text{C}$ to allow primers to bind to complementary sequences.

Extension (PCR)

Step 3 of PCR where polymerase synthesizes a new complementary strand at $72^\circ\text{C}$.

MstII

A restriction enzyme used to detect sickle cell mutations by cutting the sequence $CCTNAGG$; mutations in the hemoglobin gene remove this recognition site.

Gene therapy

A medical approach to treat disease by adding a functional gene copy, editing mutations (e.g., CRISPR-Cas9), or silencing harmful genes.

Bt Crops

Genetically modified crops containing Bacillus thuringiensis cry genes that produce insecticidal Cry proteins.

Golden Rice

Rice modified with psy and crtI genes to produce $\beta$-carotene in the endosperm to address vitamin A deficiency.

DNA Fingerprinting

A forensic technique involving PCR amplification of Short Tandem Repeats (STRs) to create a unique genetic profile.

Chymosin (rennin)

An enzyme produced via recombinant DNA technology used in over $90\%$ of global cheese production.

He Jiankui (2018)

The researcher associated with the internationally condemned first CRISPR-edited babies, raising heritable eugenics concerns.