All flashcards
Flashcard 1: What does a Southern blot specifically detect after transferring DNA to a membrane?
Answer: A specific DNA sequence using a labeled nucleic acid probe. Hybridizes probes to immobilized DNA, detecting specific sequences via autoradiography or fluorescence.
Flashcard 2: What does a Western blot specifically detect on a membrane?
Answer: A specific protein using antibodies. Uses primary and secondary antibodies to detect and quantify proteins separated by SDS-PAGE.
Flashcard 3: What is the defining feature of recombinant DNA?
Answer: 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.
Flashcard 4: What is the primary function of a restriction endonuclease in cloning?
Answer: It cleaves DNA at specific recognition sequences. Recognizes and cuts at palindromic sequences, generating fragments with sticky or blunt ends for cloning.
Flashcard 5: What is a palindromic restriction site in double-stranded DNA?
Answer: A sequence that reads the same 5′→3′ on both strands. Allows restriction enzymes to recognize and cleave symmetrically, facilitating precise DNA cutting.
Flashcard 6: What is the primary basis for separation of DNA fragments in agarose gel electrophoresis?
Answer: 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.
Flashcard 7: What does a Northern blot specifically detect on a membrane?
Answer: A specific RNA transcript using a labeled nucleic acid probe. Measures RNA abundance and size by probe hybridization after gel separation and membrane transfer.
Flashcard 8: What is the key difference between sticky ends and blunt ends after restriction digestion?
Answer: 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.
Flashcard 9: Which enzyme covalently joins DNA fragments by forming phosphodiester bonds?
Answer: DNA ligase. Catalyzes the formation of phosphodiester bonds between adjacent nucleotides, sealing nicks in the DNA backbone.
Flashcard 10: What is the function of an origin of replication (ori) on a plasmid vector?
Answer: 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.
Flashcard 11: What is the role of a selectable marker gene on a cloning vector?
Answer: It allows selection of cells that carry the vector. Confers a survival advantage, like antibiotic resistance, to identify and isolate successfully transformed cells.
Flashcard 12: Which selectable marker is most commonly used for bacterial plasmid selection?
Answer: An antibiotic resistance gene (for example, ampicillin resistance). Provides resistance to antibiotics, allowing growth of only transformed bacteria on selective media.
Flashcard 13: What is the purpose of a multiple cloning site (MCS) in a plasmid?
Answer: It provides many unique restriction sites for inserting DNA. Clusters rare-cutting restriction sites, offering flexibility for inserting foreign DNA without disrupting vector functions.
Flashcard 14: What is transformation in the context of bacterial cloning?
Answer: Uptake of exogenous DNA (often a plasmid) by bacteria. Introduces recombinant plasmids into competent bacteria, enabling propagation of cloned DNA.
Flashcard 15: What is transfection in the context of eukaryotic molecular biology?
Answer: 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.
Flashcard 16: What is the key distinction between a genomic DNA library and a cDNA library?
Answer: 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.
Flashcard 17: Which enzyme synthesizes DNA from an RNA template during cDNA library construction?
Answer: Reverse transcriptase. Uses RNA as a template to synthesize complementary DNA, capturing expressed sequences without introns.
Flashcard 18: What is the defining function of DNA polymerase in PCR?
Answer: It extends primers to synthesize new DNA strands. Adds nucleotides to the 3′-end of primers, replicating the template strand during amplification.
Flashcard 19: Which DNA polymerase is classically used in PCR because it is heat-stable?
Answer: Taq polymerase. Withstands high denaturation temperatures without losing activity, enabling automated thermal cycling.
Flashcard 20: Identify the correct order of PCR steps within one cycle.
Answer: Denaturation, annealing, extension. Separates strands, allows primer binding, and enables synthesis, exponentially amplifying DNA each cycle.
Flashcard 21: What is the approximate temperature used for DNA denaturation in PCR?
Answer: About 95∘C. Disrupts hydrogen bonds between strands, separating double-stranded DNA for primer annealing.
Flashcard 22: What is the approximate temperature range used for primer annealing in PCR?
Answer: About 50 to 65∘C. Optimizes hydrogen bonding between primers and template, ensuring specific amplification without non-specific binding.
Flashcard 23: What is the approximate temperature used for extension with Taq polymerase in PCR?
Answer: About 72∘C. Matches the optimal activity temperature of Taq polymerase for efficient nucleotide incorporation.
Flashcard 24: What is the formula for ideal PCR amplification after n cycles starting from one template?
Answer: 2n copies (ideal, assuming 100% efficiency). Represents exponential doubling per cycle, assuming perfect efficiency and no limiting factors.
Flashcard 25: Which option best explains why primers are required for PCR amplification?
Answer: 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.