IIT JAM Biotechnology flashcards

32 cards. Read the term and the definition here. The deck at the top of this page is optional.

Restriction endonuclease

Bacterial enzyme that cleaves DNA at a specific recognition sequence, usually palindromic. Type II enzymes such as EcoRI produce sticky ends used in cloning and are inhibited by host methylation.

Sticky vs blunt ends

Sticky ends are short single-stranded overhangs produced by staggered cuts; blunt ends are flush. Sticky ends base-pair with complementary overhangs and ligate far more efficiently.

Plasmid vector features

A cloning plasmid must contain an origin of replication for autonomous replication, a selectable marker such as an antibiotic-resistance gene, and a multiple cloning site for inserting foreign DNA.

Blue-white screening

Insertional inactivation of lacZα in pUC vectors. Intact lacZα gives blue colonies on X-gal plus IPTG; a cloned insert disrupts the gene, giving white recombinant colonies.

Transformation

Uptake of naked DNA by a competent bacterial cell. Competence is induced chemically with calcium chloride plus heat shock, or electrically by electroporation, then cells are plated on selective medium.

PCR primer design

Primers are short single-stranded oligonucleotides flanking the target. They anneal antiparallel to opposite strands, have similar melting temperatures, and should avoid self-complementarity and primer dimers.

Annealing temperature

The PCR step, near 50–65 °C, at which primers hybridise to template. It is set a few degrees below primer melting temperature; too low gives non-specific products, too high gives no amplification.

Taq polymerase

Heat-stable DNA polymerase from Thermus aquaticus used in PCR. It survives denaturation at about 94 °C, extends primers at about 72 °C, and lacks proofreading, so error rates are relatively high.

Reverse transcription PCR

RT-PCR converts RNA to complementary DNA using reverse transcriptase before amplification. It detects and quantifies specific transcripts; real-time RT-PCR measures expression levels fluorimetrically.

Sanger sequencing

Chain-termination method using dideoxynucleotides that lack a 3'-hydroxyl group. Incorporation stops extension; fragments separated by size reveal the sequence, typically 500–1000 bases.

Southern blotting

Transfer of size-fractionated DNA from a gel to a membrane, followed by hybridisation with a labelled probe. It detects specific sequences and is used in restriction mapping and transgene detection.

Western blotting

Separation of proteins by SDS-PAGE, transfer to a membrane, and detection with specific primary antibody plus labelled secondary antibody. It confirms protein identity, size and relative abundance.

Agarose gel electrophoresis

Technique separating DNA by size in a gel matrix under an electric field. Negatively charged DNA migrates toward the anode; smaller fragments travel farther. Fragment size is read from a ladder.

Bacterial growth curve

Plot of cell number against time: lag, exponential (log), stationary and death phases. During log phase, N = N0 × 2^(t/g), where g is the generation or doubling time.

Autoclaving

Sterilisation with saturated steam at 121 °C and 15 psi for 15–20 minutes. It denatures microbial proteins and kills spores; unsuitable for heat-labile media components or volatile chemicals.

Selectable marker

A gene carried on a vector that allows only transformed cells to grow under selective conditions, typically an antibiotic-resistance gene such as ampR or kanR used with the matching antibiotic.

cDNA library

A collection of cloned complementary DNA copies made from mRNA by reverse transcriptase. It represents only expressed genes of a tissue and lacks introns and promoter sequences.

Genomic library

A collection of clones containing fragments covering the entire genome of an organism, made by digesting genomic DNA and ligating into vectors; it includes introns and non-coding sequences.

Monoclonal antibodies

Antibodies of a single specificity produced by a hybridoma, a fusion of a B lymphocyte with an immortal myeloma cell. They are used in diagnostics, therapy and immunoassays such as ELISA.

ELISA

Enzyme-linked immunosorbent assay detecting antigen or antibody using an enzyme-conjugated reagent and chromogenic substrate. Formats include direct, indirect, sandwich and competitive; read by absorbance.

Plant tissue culture

In vitro growth of plant cells on sterile nutrient medium with auxin and cytokinin. High auxin favours roots, high cytokinin favours shoots; callus is undifferentiated dividing tissue.

Agrobacterium tumefaciens

Soil bacterium used for plant transformation. Its Ti plasmid transfers T-DNA into plant cells, integrating into the genome; the virulence genes are induced by plant phenolic compounds.

Bioprocess fermenter

A controlled vessel for large-scale microbial or cell culture, providing agitation, aeration, temperature and pH control. Stirred-tank and airlift types are common; parameters scale with oxygen demand.

Immobilised enzymes

Enzymes attached to a solid support or entrapped in a matrix, allowing reuse and continuous operation. Immobilisation often increases stability but may reduce activity through restricted substrate access.

Primary vs secondary metabolites

Primary metabolites such as ethanol, citric acid and amino acids are essential for growth and made in log phase. Secondary metabolites such as antibiotics are made mainly in stationary phase.

Biosensor

Analytical device coupling a biological recognition element (enzyme, antibody, DNA) to a transducer converting the binding event into an electrical or optical signal, as in glucose oxidase meters.

Stem cells

Cells with self-renewal capacity and the ability to differentiate into specialised types. Embryonic stem cells are pluripotent; adult stem cells are multipotent, such as haematopoietic stem cells.

RNA interference

Sequence-specific gene silencing by small interfering or microRNAs. They guide the RNA-induced silencing complex to complementary mRNA, causing cleavage or translational repression.

Site-directed mutagenesis

Technique introducing a specific base change into a cloned gene using a mismatched primer in PCR-based extension, allowing study of protein structure–function relationships.

CRISPR-Cas9

Genome-editing system in which a guide RNA directs the Cas9 nuclease to a complementary DNA sequence adjacent to a PAM, creating a double-strand break repaired by error-prone or templated pathways.

Bioinformatics BLAST

A heuristic algorithm comparing a query sequence against databases to find local alignments. Scores use match/mismatch and gap penalties; the E-value indicates expected chance matches.

Enzyme kinetics

Michaelis–Menten model: v = Vmax[S]/(Km + [S]). Km is the substrate concentration at half Vmax and reflects affinity inversely; a low Km means high affinity.