BACE DNA Extraction and Analysis 2 — Questions and Answers
Question 1: What is the role of SDS (sodium dodecyl sulfate) in a DNA extraction lysis buffer?
- It precipitates DNA out of solution for easy collection
- It denatures and solubilizes proteins, disrupts cell membranes, and helps release nucleic acids (Correct answer)
- It inhibits DNase enzymes that would degrade DNA
- It chelates magnesium ions required for membrane stability
Correct answer: It denatures and solubilizes proteins, disrupts cell membranes, and helps release nucleic acids
SDS is an anionic detergent that disrupts lipid bilayers, denatures proteins (including nucleases), and releases cellular contents including DNA.
SDS in lysis buffers disrupts lipid bilayers of cell and nuclear membranes, denatures proteins by coating them with negative charges (inactivating nucleases), and releases DNA into solution. SDS-based lysis is very effective but can interfere with downstream enzymatic steps (PCR, restriction digestion), so DNA must be purified away from SDS before use.
Question 2: In gel electrophoresis of DNA, what determines the direction and rate of DNA migration?
- DNA migrates toward the positive electrode (anode) because it is negatively charged; smaller fragments migrate faster (Correct answer)
- DNA migrates toward the negative electrode because it is positively charged; larger fragments migrate faster
- DNA migrates based on base composition (GC content) regardless of size
- All DNA fragments of the same base pair length migrate at exactly the same speed regardless of gel percentage
Correct answer: DNA migrates toward the positive electrode (anode) because it is negatively charged; smaller fragments migrate faster
DNA has a uniform negative charge from its phosphate backbone and migrates toward the positive anode; smaller fragments move faster through the gel matrix than larger ones.
The phosphate backbone of DNA gives it a uniform negative charge-to-mass ratio, so all DNA fragments migrate toward the anode at a rate governed primarily by molecular size through the agarose gel matrix. Larger fragments are retarded more by pore interactions; smaller fragments pass through pores more easily. Log(size) is proportional to migration distance when compared to a DNA ladder.
Question 3: What is the purpose of adding ethanol during a silica membrane DNA purification protocol (spin column)?
- To digest proteins and cellular debris that would clog the membrane
- To bind DNA to the silica membrane by reducing aqueous solvation of DNA (Correct answer)
- To elute purified DNA from the column for downstream use
- To denature double-stranded DNA into single strands for PCR
Correct answer: To bind DNA to the silica membrane by reducing aqueous solvation of DNA
Ethanol (typically with a chaotropic salt wash buffer) reduces the hydration shell around DNA molecules, promoting their adsorption to the negatively charged silica membrane.
Silica membrane columns work via selective adsorption: in high-chaotrope, high-ethanol conditions, DNA loses its water solvation shell and adsorbs to the silica surface. RNA and proteins are not retained or are washed away. After binding and washing, pure DNA is eluted with low-salt buffer or water, which restores hydration and releases DNA from silica.
Question 4: A DNA sample shows an A260/A280 ratio of 1.5 when measured by spectrophotometry. What does this indicate?
- The DNA is very pure with no protein contamination
- The DNA sample is likely contaminated with protein, which absorbs strongly at 280 nm (Correct answer)
- The sample contains RNA but no protein contamination
- The concentration of DNA is too low to measure accurately
Correct answer: The DNA sample is likely contaminated with protein, which absorbs strongly at 280 nm
Pure double-stranded DNA has an A260/A280 ratio of ~1.8; a ratio of 1.5 indicates protein contamination since proteins absorb at 280 nm, reducing this ratio.
The A260/A280 ratio is a standard DNA purity metric. Pure dsDNA: ~1.8; pure RNA: ~2.0. Aromatic amino acids in proteins absorb at 280 nm. Protein contamination raises A280, lowering the ratio below 1.8. A ratio of 1.5 indicates significant protein carryover — this can inhibit downstream enzymes. The A260/A230 ratio (should be >1.7-2.2) detects contamination with carbohydrates, salts, EDTA, or phenol.
Question 5: What is restriction fragment length polymorphism (RFLP) analysis used for?
- Amplifying specific DNA sequences using thermal cycling
- Identifying DNA sequence differences between individuals by digesting DNA with restriction enzymes and comparing fragment sizes (Correct answer)
- Sequencing the complete genome of an organism
- Measuring the concentration of DNA in a solution
Correct answer: Identifying DNA sequence differences between individuals by digesting DNA with restriction enzymes and comparing fragment sizes
RFLP analysis detects DNA sequence variations by using restriction enzymes to cut DNA at specific recognition sites; sequence differences create different fragment size patterns on gels.
Restriction enzymes cut DNA at specific palindromic sequences. Single nucleotide polymorphisms can create or destroy restriction sites, causing DNA from different individuals to produce fragments of different sizes that separate differently on gels. RFLP was historically used for genetic mapping, disease diagnosis, paternity testing, and forensics, but has largely been superseded by PCR-based methods.
Question 6: Why is EDTA (ethylenediaminetetraacetic acid) often included in DNA storage and elution buffers (TE buffer)?
- It increases DNA solubility in aqueous solutions
- It chelates divalent metal ions (Mg2+, Mn2+) required by DNase enzymes, preventing DNA degradation (Correct answer)
- It acts as an antifungal agent to prevent microbial growth in DNA stocks
- It provides a reducing environment to prevent DNA oxidation
Correct answer: It chelates divalent metal ions (Mg2+, Mn2+) required by DNase enzymes, preventing DNA degradation
EDTA chelates Mg2+ and other divalent cations required as cofactors by DNase enzymes, inhibiting their activity and protecting stored DNA from degradation.
TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) is the standard DNA storage buffer. Tris provides pH buffering. EDTA at 1 mM is sufficient to chelate Mg2+ ions needed by DNase enzymes. However, EDTA also inhibits polymerases, so DNA to be used in PCR must be diluted substantially or stored in nuclease-free water. DNA stored in TE at -20°C remains stable for years.
BACE DNA Extraction and Analysis 2
What is the role of SDS (sodium dodecyl sulfate) in a DNA extraction lysis buffer?