Biotechnology Exam 2 Methods Map Review

Comprehensive vocabulary flashcards covering recombinant DNA techniques, PCR, sequencing technologies, hybridization assays, upstream protein expression, and downstream purification methods from the Exam 2 Methods Map.

Recombinant DNA

A DNA molecule formed by joining DNA fragments from two different sources together into a single molecule.

Restriction Enzyme

An enzyme that recognizes a specific target DNA sequence and cleaves the DNA duplex at or near that site, cutting around the gene of interest rather than within it.

Sticky Ends

Short, single-stranded complementary overhangs generated on DNA fragments by restriction enzyme cleavage that can base-pair with matching ends.

DNA Ligase

An enzyme that catalytically joins compatible paired DNA fragments by sealing the phosphodiester bonds of the sugar-phosphate backbone.

Multiple Cloning Site (MCS)

A designated region within a plasmid vector containing multiple unique restriction enzyme recognition sites designed to receive inserted DNA.

pUC19

A standard cloning plasmid vector measuring $2,686\,\text{base pairs}$ in length that features an ampicillin-resistance gene, a pMB1 origin of replication, and an MCS positioned inside the lacZ gene.

cDNA (Complementary DNA)

DNA synthesized from a mature mRNA template using reverse transcriptase; it contains an intron-free coding sequence suitable for expressing eukaryotic proteins in bacterial hosts.

TA Cloning

A cloning method pairing the single adenine (A) overhangs added to the 3-prime ends of PCR products by Taq polymerase with the complementary single thymine (T) overhangs of a linearized T vector.

Chemical Transformation

A plasmid delivery procedure where competent bacterial cells are treated with $CaCl_2$ to neutralize cell-surface charge, kept on ice for approximately $30\,\text{minutes}$, and exposed to a heat shock at approximately $42\,^\circ\text{C}$ for $30\,\text{seconds}$.

Electroporation

A physical transformation method that applies a brief high-voltage electrical pulse (approximately $1,200\,\text{V}$ for $3\,\text{ms}$) to generate transient openings in the cell membrane for plasmid entry.

Blue-White Screening

A visual screening technique using X-gal and the lacZ gene, where recombinant plasmids with disrupted lacZ produce white colonies, whereas non-recombinant vectors with intact beta-galactosidase activity produce blue colonies.

Genomic Library

A comprehensive collection of cloned DNA fragments representing an organism's entire chromosomal DNA, including coding exons, introns, regulatory elements, and noncoding sequences.

cDNA Library

A cloned collection of DNA sequences synthesized from a tissue's spliced mRNA population, capturing only expressed genes without introns.

Colony Hybridization

A screening technique that transfers colonies from a master plate onto a membrane, lyses cells with NaOH to denature DNA, and uses a labeled complementary single-stranded probe to locate the bacterial clone bearing the target gene.

Polymerase Chain Reaction (PCR)

An in vitro DNA amplification technique that repeatedly cycles through high-temperature denaturation ($94\text{--}96\,^\circ\text{C}$), primer annealing ($52\text{--}58\,^\circ\text{C}$), and enzymatic extension ($72\,^\circ\text{C}$) to yield theoretical copies equal to $\text{starting copies} \times 2^N$.

Taq DNA Polymerase

A heat-stable DNA polymerase isolated from Thermus aquaticus that requires $Mg^{2+}$ ions as a cofactor to synthesize new complementary DNA strands at $72\,^\circ\text{C}$.

Primer Melting Temperature ($T_m$)

The predicted temperature governing primer annealing, calculated using the formula $T_m = 4(G + C) + 2(A + T)\,^\circ\text{C}$.

Sanger Sequencing

First-generation sequencing that uses a single primer, DNA polymerase, standard dNTPs, and chain-terminating dideoxynucleotides (ddNTPs) lacking a 3-prime hydroxyl group to establish the base sequence.

Electropherogram

A colored four-dye peak trace generated by laser detection of fluorescent fragments during capillary automated Sanger sequencing.

Emulsion PCR

An amplification step used in second-generation sequencing where individual DNA fragments attached to beads are clonally amplified inside isolated aqueous oil droplets.

Roche 454 Pyrosequencing

A second-generation sequencing platform reading $200\text{--}300\,\text{nucleotides}$ by converting released pyrophosphate (PPi) into ATP via ATP sulfurylase, which fuels a luciferase reaction to produce detectable light.

Ion Torrent Sequencing

A second-generation semiconductor sequencing method reading $400\text{--}600\,\text{nucleotides}$ by measuring the release of hydrogen ions ($H^+$) and the resulting pH change upon nucleotide incorporation.

Nanopore Sequencing

A third-generation single-molecule sequencing technology (e.g., MinION) that measures characteristic disruptions in ionic current as intact native DNA strands thread through a nanoscale protein pore.

Sequencing Coverage

A numerical metric representing the average number of distinct sequencing reads that align to and overlap a specific nucleotide position in a reference sequence.

Southern Blotting

A molecular assay detecting specific DNA restriction fragments via agarose gel separation, capillary alkaline transfer to a membrane, and hybridization with a labeled single-stranded DNA probe.

Northern Blotting

An expression assay that separates isolated RNA by size on a gel, transfers it to a membrane, and hybridizes with a labeled probe to determine transcript size and approximate abundance relative to a housekeeping gene.

RT-PCR (Reverse-Transcription PCR)

An endpoint gene-expression detection method where reverse transcriptase first converts target mRNA into cDNA, which is subsequently amplified by PCR and analyzed on an agarose gel.

qPCR (Real-Time PCR)

A quantitative PCR assay that monitors fluorophore emissions during every cycling stage to measure target amplification in real time.

Threshold Cycle ($C_t$)

The cycle number in real-time PCR at which the reporter fluorescence crosses an established threshold; lower values reflect higher starting target concentrations.

SYBR Green

A low-cost qPCR dye that fluoresces upon non-specific intercalation into double-stranded DNA, capable of binding both target products and unwanted off-target duplexes.

TaqMan Probe

A sequence-specific qPCR oligonucleotide labeled with a 5-prime reporter fluorophore and a 3-prime quencher that fluoresces when cleaved by polymerase exonuclease activity.

Fluorescence In Situ Hybridization (FISH)

A cytogenetic hybridization technique that utilizes fluorescently labeled nucleic acid probes directly on preserved cells or tissue slices to map chromosomal coordinates or visualize localized cellular RNA expression.

Upstream Processing

The phase of biotechnology manufacturing encompassing host selection, culture growth in fermenters or bioreactors, and the biochemical induction of recombinant gene expression.

Bioreactor

An aerobic culture vessel fitted with impellers, spargers, probes, and cooling jackets that regulates temperature, dissolved oxygen, carbon dioxide, and pH to grow cells producing recombinant proteins.

IPTG

A non-metabolizable lactose analog added during peak cell growth to displace the lac repressor protein from the operator, thereby inducing transcription of genes downstream of the lac promoter.

Directed Molecular Evolution

An iterative protein engineering method utilizing mutagenic PCR to generate random gene variants, followed by functional activity assays to isolate variants with improved catalytic properties.

Site-Directed Mutagenesis

A targeted protein engineering technique used to introduce a specific, predetermined nucleotide modification into a known gene sequence.

Downstream Processing

The operational stages following protein expression dedicated to cell lysis or harvesting, crude extract stabilization, separation, chromatography purification, and final formulation.

Dialysis

A buffer exchange and descaling method where a protein solution inside semipermeable membrane tubing (such as SnakeSkin tubing) is placed in low-salt buffer to allow passive diffusion of small salts, solvents, and additives.

Size-Exclusion Chromatography

A separation method using columns packed with porous beads, whereby large proteins elute first because they are excluded from the pores while smaller proteins travel through pore channels and elute later.

Ion-Exchange Chromatography

A chromatographic technique separating proteins according to surface charge, utilizing positive anion-exchange resins or negative cation-exchange resins, with bound proteins eluted by raising the salt gradient.

Isoelectric Point ($pI$)

The specific pH at which a protein exhibits a net zero electrical charge; proteins carry net positive charge when buffer pH is below their $pI$ and net negative charge when buffer pH is above their $pI$.

Affinity Chromatography

A highly specific purification strategy exploiting reversible non-covalent binding between an immobilized column ligand (such as maltose) and a specialized tag fused to the target protein.

Hydrophobic-Interaction Chromatography (HIC)

A purification method that binds proteins via exposed hydrophobic surface regions at high salt concentrations and elutes them as the salt concentration is lowered.

Two-Dimensional (2D) Gel Electrophoresis

A high-resolution protein separation method combining isoelectric focusing along a pH gradient in the first dimension (sorting by $pI$) with SDS-PAGE in the second dimension (sorting by molecular mass).