IIT JAM Biotechnology key terms

An enzyme that cuts double-stranded DNA at a specific recognition sequence, often leaving sticky or blunt ends. Bacteria use these enzymes as a defence against foreign DNA, and molecular biologists use them to cut and join DNA fragments.

A DNA sequence that reads the same on the complementary strand when both are read in the 5' to 3' direction, such as GAATTC. Most type II restriction enzymes recognise such sequences, which is why they cut both strands at symmetric positions.

A short single-stranded overhang produced when a restriction enzyme makes staggered cuts in DNA. Complementary overhangs on two different fragments can base-pair, allowing them to be joined efficiently by DNA ligase.

The joining of two DNA molecules by forming phosphodiester bonds between adjacent nucleotides, catalysed by DNA ligase. In cloning it seals a vector to an insert, and it also seals nicks during DNA replication and repair.

A small, self-replicating DNA molecule used to carry a foreign DNA insert into a host cell. It must have an origin of replication, a selectable marker and a unique restriction site for inserting the fragment.

A small circular extrachromosomal DNA molecule found in many bacteria that replicates independently of the chromosome. Engineered plasmids are the most common cloning vectors and can also carry genes for expressing recombinant proteins.

A gene carried on a vector that lets host cells containing the vector survive or be detected under specific conditions, for example an antibiotic-resistance gene. It is essential for identifying transformed cells among a large background of non-transformants.

The uptake of free DNA from the environment by a competent bacterial cell, which may then express the new genes. In the laboratory it is induced artificially, for example by calcium chloride and heat shock or by electroporation, to introduce plasmids.

A bacterial cell made able to take up external DNA, usually by chemical treatment with divalent cations or by brief high-voltage pulses. Competence is a physiological state that allows DNA to cross the cell envelope.

A technique that applies short high-voltage pulses to cells, transiently making their membranes permeable so that DNA or other molecules can enter. It is used for bacteria, yeast, plant protoplasts and mammalian cells.

The introduction of foreign nucleic acid into eukaryotic cells, typically cultured animal cells, using methods such as liposome-mediated delivery, electroporation or calcium phosphate precipitation. The term usually excludes viral infection, which is called transduction.

The transfer of bacterial DNA from one cell to another by a bacteriophage particle. In gene therapy the term also describes delivery of genetic material to eukaryotic cells using a viral vector.

A virus that infects bacteria, attaching to a specific host receptor and injecting its nucleic acid. Phage lambda and M13 are widely used as cloning vectors and phage display systems.

An in vitro method that amplifies a specific DNA region through repeated cycles of denaturation, primer annealing and extension by a thermostable DNA polymerase. Each cycle roughly doubles the target, giving exponential accumulation of product.

A short single-stranded oligonucleotide that provides a free 3'-hydroxyl group for a DNA polymerase to extend. In PCR, two primers flanking the target define the region that is amplified.

The temperature at which PCR primers hybridise to their complementary target sequences, usually a few degrees below the primer melting temperature. It controls specificity: too low gives non-specific products, too high gives little or no amplification.

A heat-stable DNA polymerase originally isolated from the thermophilic bacterium Thermus aquaticus. Its ability to survive the high denaturation temperature of PCR cycles made automated amplification practical.

Synthesis of a complementary DNA strand from an RNA template, catalysed by reverse transcriptase. It is the key step in converting mRNA into cDNA for cloning, library construction and quantitative PCR.

An RNA-dependent DNA polymerase that copies RNA into DNA and also has ribonuclease H activity. Retroviruses encode it, and engineered forms are used to make cDNA from mRNA in the laboratory.

A collection of cloned DNA fragments generated by reverse transcription of the mRNA population of a cell or tissue. It represents only expressed genes, unlike a genomic library, which contains all DNA including non-coding regions.

A set of cloned DNA fragments that together represent the entire genome of an organism, made by cutting genomic DNA and inserting the fragments into vectors. It includes coding and non-coding sequences and is independent of gene expression.

A technique in which DNA fragments separated by gel electrophoresis are transferred to a membrane and detected with a labelled complementary probe. It reveals the presence and size of specific sequences in a complex sample.

A method in which proteins separated by gel electrophoresis are transferred to a membrane and detected with antibodies specific to the target protein. It is used to confirm protein identity, size and relative abundance.

The base-pairing of two complementary single-stranded nucleic acid molecules to form a double-stranded duplex. Labelled probes exploit it to detect specific DNA or RNA sequences in blots, colonies and in situ preparations.

An antibody of a single specificity produced by a clone of hybridoma cells formed by fusing a B lymphocyte with a myeloma cell. Because every molecule binds the same epitope, it gives highly reproducible detection and is widely used in diagnostics and therapy.

An immortal cell line created by fusing an antibody-secreting B cell with a myeloma cell. It combines the antibody specificity of the lymphocyte with the unlimited division of the tumour cell, allowing continuous monoclonal antibody production.

A plate-based immunoassay in which an enzyme conjugated to an antibody converts a substrate to a coloured or fluorescent product, giving a signal proportional to antigen concentration. It is used for quantitative detection of hormones, antigens and antibodies.

A DNA sequencing method that uses chain-terminating dideoxynucleotides to produce fragments of different lengths, which are separated by size to read the sequence. It remains the standard for verifying individual sequences.

Massively parallel sequencing technologies that read millions of DNA fragments simultaneously, producing short reads at low cost per base. Reads are then aligned to a reference or assembled de novo to reconstruct genomes or transcriptomes.

The application of computational methods to store, retrieve and analyse biological data such as sequences, structures and expression profiles. It includes sequence alignment, database searching, phylogenetics and structural prediction.

A vessel in which microorganisms, animal cells or enzymes are cultured under controlled conditions of temperature, pH, aeration and nutrient supply to produce a desired product. It may operate in batch, fed-batch or continuous mode.

In industrial biotechnology, the large-scale cultivation of microorganisms or cells to convert substrates into products such as antibiotics, organic acids or recombinant proteins. In a narrower biochemical sense it means energy generation without an external electron acceptor.

An enzyme attached to or entrapped in an insoluble support so that it can be reused and easily separated from the reaction mixture. Immobilisation often increases stability and allows continuous operation in industrial processes.

The study of reaction rates catalysed by enzymes and how they depend on substrate concentration, inhibitors, pH and temperature. Parameters such as Km and Vmax describe substrate affinity and maximum catalytic capacity.

A reversible inhibition in which the inhibitor binds the enzyme's active site, competing with the substrate. It raises the apparent Km but leaves Vmax unchanged, because inhibition can be overcome by increasing substrate concentration.

Control of enzyme activity by a molecule binding at a site remote from the active site and changing the enzyme's conformation. It produces sigmoidal kinetics and is central to metabolic pathway control and feedback inhibition.

A genetically programmed form of cell death marked by chromatin condensation, DNA fragmentation and membrane blebbing without inflammation. It removes damaged or unwanted cells during development and tissue homeostasis.

A family of cysteine proteases that cleave proteins after aspartate residues and execute apoptosis. Initiator caspases become active in response to death signals and activate effector caspases that dismantle the cell.

Cell death caused by severe injury, infection or ischaemia, characterised by swelling, membrane rupture and release of cellular contents that provoke inflammation. It is largely uncontrolled, unlike apoptosis.

A control point where the cell verifies that conditions are suitable before proceeding to the next phase, such as the G1/S and G2/M transitions. Checkpoint failure allows propagation of DNA damage and contributes to cancer.

A gene whose gain-of-function mutation or overexpression drives uncontrolled cell proliferation and contributes to cancer. Many oncogenes encode growth factors, receptors, signalling kinases or transcription factors.

A gene that normally restrains cell division or promotes repair and apoptosis, so that loss of function predisposes to cancer. Both alleles usually must be inactivated, consistent with the two-hit model.