Recombinant DNA and Biotechnology (1B) - MCAT Biological and Biochemical Foundations of Living Systems

A specific protein using antibodies. Uses primary and secondary antibodies to detect and quantify proteins separated by SDS-PAGE.

DNA constructed by joining sequences from different sources. Combines genetic material from multiple organisms or sources to create novel DNA molecules for cloning or expression.

It cleaves DNA at specific recognition sequences. Recognizes and cuts at palindromic sequences, generating fragments with sticky or blunt ends for cloning.

A sequence that reads the same $5'\to 3'$ on both strands. Allows restriction enzymes to recognize and cleave symmetrically, facilitating precise DNA cutting.

Fragment size; smaller fragments migrate farther toward the anode. Applies an electric field where negatively charged DNA moves through the gel matrix, with smaller pieces traveling faster.

A specific RNA transcript using a labeled nucleic acid probe. Measures RNA abundance and size by probe hybridization after gel separation and membrane transfer.

Sticky ends have ss overhangs; blunt ends have no overhangs. Sticky ends enable complementary base pairing for efficient ligation, while blunt ends require direct joining.

DNA ligase. Catalyzes the formation of phosphodiester bonds between adjacent nucleotides, sealing nicks in the DNA backbone.

It enables plasmid replication in the host cell. Serves as the starting point for DNA replication machinery, ensuring the plasmid is copied during cell division.

It allows selection of cells that carry the vector. Confers a survival advantage, like antibiotic resistance, to identify and isolate successfully transformed cells.

An antibiotic resistance gene (for example, ampicillin resistance). Provides resistance to antibiotics, allowing growth of only transformed bacteria on selective media.

It provides many unique restriction sites for inserting DNA. Clusters rare-cutting restriction sites, offering flexibility for inserting foreign DNA without disrupting vector functions.

Uptake of exogenous DNA (often a plasmid) by bacteria. Introduces recombinant plasmids into competent bacteria, enabling propagation of cloned DNA.

Introduction of nucleic acids into eukaryotic cells. Delivers DNA or RNA into animal or plant cells using methods like electroporation or liposomes for gene expression studies.

Genomic: all DNA; cDNA: reverse-transcribed mRNA (no introns). Genomic libraries include introns and non-coding regions, while cDNA libraries represent expressed genes for protein studies.

Reverse transcriptase. Uses RNA as a template to synthesize complementary DNA, capturing expressed sequences without introns.

It extends primers to synthesize new DNA strands. Adds nucleotides to the $3'$-end of primers, replicating the template strand during amplification.

Taq polymerase. Withstands high denaturation temperatures without losing activity, enabling automated thermal cycling.

Denaturation, annealing, extension. Separates strands, allows primer binding, and enables synthesis, exponentially amplifying DNA each cycle.

About $95,^{\circ}\text{C}$. Disrupts hydrogen bonds between strands, separating double-stranded DNA for primer annealing.

About $50$ to $65,^{\circ}\text{C}$. Optimizes hydrogen bonding between primers and template, ensuring specific amplification without non-specific binding.

About $72,^{\circ}\text{C}$. Matches the optimal activity temperature of Taq polymerase for efficient nucleotide incorporation.

$2^n$ copies (ideal, assuming $100%$ efficiency). Represents exponential doubling per cycle, assuming perfect efficiency and no limiting factors.

DNA polymerase cannot initiate synthesis de novo; it needs a $3'$-OH. Provides the necessary $3'$-OH group for DNA polymerase to extend, as it requires an existing chain to build upon.