DNA Extraction and Analysis Flashcards
6 cards from real BACE practice questions. Tap to flip, then mark Knew It or Still Learning — missed cards come back until you master them.
Read the first 6 DNA Extraction and Analysis flashcards as text
In a PCR reaction, what is the function of the denaturation step?
Answer: It separates the double-stranded DNA template into two single strands for primer binding
Denaturation at 94-98°C breaks the hydrogen bonds between complementary base pairs, separating the double helix into single-stranded templates that primers can bind.
What is the function of a DNA ladder (molecular weight marker) in gel electrophoresis?
Answer: It provides a set of DNA fragments of known sizes to estimate the size of unknown fragments
A DNA ladder contains multiple DNA fragments of precisely known sizes, allowing estimation of unknown fragment sizes by comparing their migration distance to the ladder pattern.
Which technique allows amplification of a specific DNA sequence from a complex mixture using only two oligonucleotide primers?
Answer: Polymerase Chain Reaction (PCR)
PCR uses two primers flanking the target sequence and thermostable DNA polymerase to exponentially amplify a specific region through repeated cycles of denaturation, annealing, and extension.
What problem arises when PCR primers have a high degree of self-complementarity?
Answer: Primers can fold back on themselves forming hairpin structures or anneal to each other forming primer dimers reducing PCR efficiency
Self-complementary primer sequences form intramolecular hairpins or intermolecular primer dimers that reduce the concentration of free primers available to bind the template.
What is the significance of the melting temperature (Tm) of a PCR primer?
Answer: It is the temperature at which 50% of primers are annealed to complementary template used to set the annealing temperature
Tm is the temperature at which half the primer molecules are annealed to their complementary template; the PCR annealing temperature is typically set 3-5°C below Tm for optimal specificity.
When performing a restriction enzyme digest, why is it important to use the buffer recommended by the enzyme manufacturer?
Answer: Restriction enzymes require specific salt, pH, and cofactor conditions for optimal activity and cleavage specificity
Each restriction enzyme has optimal activity and specificity within a narrow range of salt concentration, pH, and cofactors (Mg2+); incorrect buffer conditions reduce activity and may cause 'star activity' (non-specific cleavage).