q4 final biotech correct (copy)

Q: How many species have been named in the last 250 years?

A: 2 million

Q: What are two differences between DNA and RNA?

A: DNA contains thymine and deoxyribose; RNA contains uracil and ribose.

Q: What is true about transcription?

A: Transcription makes RNA from DNA and occurs in the nucleus.

Q: What is the importance of the CO1 gene?

A: It encodes a protein critical to the electron transport chain.

Q: What accounts for higher interspecies variability in the CO1 sequence?

A: Degeneracy of the genetic code.

Q: What nucleotide position is most impactful when changed?

A: 1st position.

Q: If the expected nucleotide variation is 2%, how many differences will there be in 600 bp?

A: 12 differences.

Q: Why is it important that CO1 has regions of variability and conservation?

A: Variability distinguishes species; conservation allows primer binding.

Q: What does Proteinase K do?

A: Cleaves peptide bonds in proteins to help release DNA.

Q: What is the purpose of RNase A?

A: To digest RNA during DNA purification.

Q: How do we obtain the CO1 sequence?

A: Extract DNA, PCR amplify CO1, purify, and sequence.

Q: What charge does the silica matrix have?

A: Neutral (but binds DNA via Na⁺ cation bridge in high salt).

Q: Why does DNA bind to the silica column?

A: Na⁺ ions create a cation bridge between negatively charged DNA and silica.

Q: What is the purpose of lysis in DNA extraction?

A: To break open cells and release DNA.

Q: What is the purpose of the binding step?

A: To attach DNA to the silica membrane in the spin column.

Q: What is the purpose of the wash step?

A: To remove proteins, salts, and other impurities.

Q: What is the purpose of the elution step?

A: To release purified DNA from the silica column using water or buffer.

Q: Why does DNA migrate toward the positive electrode in electrophoresis?

A: DNA is negatively charged due to its phosphate backbone.

Q: What creates the wells in an agarose gel?

A: A sample comb.

Q: What is the purpose of loading buffer in gel electrophoresis?

A: To weigh down the sample and track migration with dyes.

Q: Bromophenol Blue migrates at the same rate as what size DNA fragment?

A: ~300 base pairs.

Q: Why is ethidium bromide useful for visualizing DNA?

A: It intercalates between DNA base pairs and fluoresces under UV light.

Q: What is the purpose of the wash step in DNA extraction?

A: To remove unbound contaminants from the column.

Q: What is the purpose of the elution step in DNA extraction?

A: To release purified DNA from the silica column.

Q: Why does DNA migrate during electrophoresis?

A: DNA is negatively charged and moves toward the positive electrode.

Q: What is used to create wells in an agarose gel?

A: Sample combs.

Q: What is the purpose of loading buffer?

A: It helps the DNA sink and contains tracking dye like bromophenol blue.

Q: Bromophenol Blue migrates with what size DNA fragment in a 1% gel?

A: Approximately 300 base pairs.

Q: Why is ethidium bromide (EtBr) useful for visualizing DNA?

A: It intercalates into DNA and fluoresces under UV light.

Q: What is the purpose of running a gel with gDNA?

A: To verify the presence and quality of genomic DNA.

Q: PCR is based on what natural process?

A: DNA replication.

Q: During which phase of the cell cycle does DNA replication occur?

A: S phase.

Q: What is needed for PCR?

A: Template DNA, DNA polymerase, primers, dNTPs, and buffer.

Q: What are the steps of PCR?

A: Denaturation, annealing, and extension.

Q: What is the expected size of the CO1 amplicon?

A: Approximately 650 base pairs.

Q: Why run a DNA ladder on a gel?

A: For size comparison.

Q: True or false: the forward primer generates the top strand.

A: True.

Q: Why are ddNTPs useful in sequencing?

A: They terminate synthesis because they lack a 3' hydroxyl group.

Q: What happens if you use only ddNTPs in a sequencing reaction?

A: You’d get only 1 bp fragments; you need both dNTPs and ddNTPs for a ladder of different lengths.

Q: What is the first step in sequence editing?

A: Trimming low-quality bases from the ends.

Q: Why are sequence quality scores low at the ends?

A: Due to poor resolution of short DNA fragments.

Q: What does the forward sequencing reaction generate?

A: The sense strand of CO1.

Q: What does the "N" represent in a DNA sequence?

A: An ambiguous nucleotide (uncertain base).

Q: What is a contig?

A: A continuous sequence assembled from overlapping reads.

Q: What are the stop codons in mitochondrial DNA?

A: UAA, UAG, AGA, AGG.

Q: What indicates a sequencing error when evaluating CO1?

A: A stop codon appearing in all three reading frames.

Q: What is the importance of DNA barcoding?

A: Identifying species, conserving biodiversity, and monitoring ecosystems.

Q: What does iBOL stand for?

A: International Barcode of Life project.

Q: What is BOLD?

A: Barcode of Life Database, used for storing barcode sequences.

Q: What is the process of barcoding bees?

A: Collect samples, extract DNA, PCR amplify CO1, sequence.

Q: What are proteins made of?

A: Amino acids.

Q: What is not a characteristic of amino acid side chains?

A: Cytotoxicity (this is a property of substances, not side chains themselves).

Q: Where are the instructions to make CO1 found?

A: Mitochondrial DNA (mtDNA).

Q: What are the bonds within and between DNA strands?

A: Phosphodiester bonds link nucleotides; hydrogen bonds link complementary strands.

Q: What is meristics?

A: A traditional method of identifying species based on physical traits.

Q: What are the categories of amino acids?

A: Basic, nonpolar/hydrophobic, polar uncharged, and acidic.

Q: How do proteins form?

A: By linking amino acids with peptide (covalent) bonds and releasing a water molecule (condensation reaction).

Q: What is DNA → RNA called, and where does it occur?

A: Transcription; occurs in the nucleus.

Q: What is RNA → protein called, and where does it occur?

A: Translation; occurs in the ribosome.

Q: Where does RNA polymerase bind to start transcription?

A: Promoter regions on the DNA.

Q: Why was CO1 chosen for DNA barcoding?

A: It’s a protein-coding gene that is conserved across species yet variable enough to distinguish between them.

Q: Why was mitochondrial DNA (mtDNA) chosen for barcoding?

A: It has high copy number, no introns, and more genetic differences between species.

Q: What organism is agarose derived from?

A: Red algae, specifically Gracilaria.

Q: At what wavelength is ethidium bromide (EtBr) excited?

A: Around 300 nm.

Q: At what wavelength does EtBr emit light?

A: Around 600 nm.

Q: Why must DNA be isolated after PCR before sequencing?

A: Residual PCR components can reduce sequencing quality.

Q: Why do ddNTPs terminate synthesis in sequencing?

A: They lack a 3' hydroxyl group, preventing addition of more nucleotides.

Q: What does an electropherogram represent?

A: The raw output of a DNA sequencing reaction.

Q: What does each peak on an electropherogram represent?

A: A nucleotide (base) in the sequence.

Q: How are sequencing quality scores determined?

A: By the shape and resolution of the peaks on the electropherogram.

Q: What is a drawback of the Sanger (chain termination) method of sequencing?

A: It is inefficient for samples with many species; separating sequences is labor-intensive and costly.