BIOL 235 Midterm 2 Exam REVIEW 2026/2027
MOST RECENT QUESTIONS AND SOLUTIONS
• What is the starting point for DNA sequencing? -✓✓ A biological sample such as
tissue, bacterial culture, or environmental sample.
• What must be done to DNA/RNA after obtaining a sample? -✓✓ It must be extracted
immediately or stored promptly.
• What is the purpose of reverse transcription in RNA extraction? -✓✓ To convert RNA
into cDNA.
• What are the two main paths for sequencing strategy? -✓✓ Path 1: Clone library; Path
2: PCR amplification.
• What is required for Sanger sequencing? -✓✓ Many copies of a single purified DNA
fragment.
• What is the core principle of Sanger sequencing? -✓✓ Chain termination using
dideoxynucleotides (ddNTPs).
• What happens when a ddNTP is incorporated during Sanger sequencing? -✓✓ The
chain cannot extend, causing termination.
• What is the difference between old and modern Sanger sequencing? -✓✓ Old Sanger
used one tube with all ddNTPs; modern Sanger uses four separate tubes for each
nucleotide.
• What method is used to read modern Sanger sequencing results? -✓✓ Capillary
electrophoresis and automatic laser detection.
• How are peaks in a chromatogram analyzed? -✓✓ Each peak represents one base,
with colors corresponding to nucleotides (A = green, T = red, G = black, C = blue).
• What are the limitations of Sanger sequencing? -✓✓ Can only read ~500 nt reliably
and is not suitable for metagenomic samples.
• What is the first step in analyzing a chromatogram? -✓✓ Identify clear, non-
overlapping peaks.
• What is the purpose of using BLAST after sequencing? -✓✓ To determine taxonomy
by comparing the sequence to a nucleotide database.
, • What are the modifications required for DNA fragments before sequencing by
synthesis? -✓✓ Adapters, indexes (barcodes), and primer binding sites.
• What is the advantage of sequencing by synthesis (Illumina)? -✓✓ Very high accuracy
(~99%) and massively parallel sequencing.
• What is a disadvantage of nanopore sequencing? -✓✓ Lower accuracy (~92-97%)
compared to other methods.
• What is the purpose of using specific genes in PCR? -✓✓ To detect one species,
strain, or functional gene.
• What are universal genes used for in PCR? -✓✓ To amplify all members of a large
group of organisms.
• What is the role of conserved regions in universal genes? -✓✓ They are where
universal primers bind.
• What is the purpose of hypervariable regions in universal genes? -✓✓ Used to identify
organisms.
• What is the output format of Illumina sequencing? -✓✓ Two FASTQ files per sample
(forward + reverse reads).
• What is a key feature of nanopore sequencing technology? -✓✓ It allows real-time
sequencing without PCR.
• What is the significance of the E-value in BLAST results? -✓✓ It indicates the
statistical significance of the match.
• What does a high % identity in BLAST results indicate? -✓✓ It identifies the species
with high confidence.
• What is the purpose of trimming low-quality ends in chromatogram analysis? -✓✓ To
remove fuzzy, overlapping peaks for accurate sequence determination.
• What is the function of indexes (barcodes) in sequencing? -✓✓ To identify which
sample fragments came from during multiplexing.
• What is bridge amplification in Illumina sequencing? -✓✓ PCR amplification that
occurs directly on the flow cell to create clusters of identical DNA fragments.
• How does Nanodrop quantify DNA? -✓✓ It measures UV absorbance at 260 nm to
assess nucleic acids and reports total nucleic acid concentration.
MOST RECENT QUESTIONS AND SOLUTIONS
• What is the starting point for DNA sequencing? -✓✓ A biological sample such as
tissue, bacterial culture, or environmental sample.
• What must be done to DNA/RNA after obtaining a sample? -✓✓ It must be extracted
immediately or stored promptly.
• What is the purpose of reverse transcription in RNA extraction? -✓✓ To convert RNA
into cDNA.
• What are the two main paths for sequencing strategy? -✓✓ Path 1: Clone library; Path
2: PCR amplification.
• What is required for Sanger sequencing? -✓✓ Many copies of a single purified DNA
fragment.
• What is the core principle of Sanger sequencing? -✓✓ Chain termination using
dideoxynucleotides (ddNTPs).
• What happens when a ddNTP is incorporated during Sanger sequencing? -✓✓ The
chain cannot extend, causing termination.
• What is the difference between old and modern Sanger sequencing? -✓✓ Old Sanger
used one tube with all ddNTPs; modern Sanger uses four separate tubes for each
nucleotide.
• What method is used to read modern Sanger sequencing results? -✓✓ Capillary
electrophoresis and automatic laser detection.
• How are peaks in a chromatogram analyzed? -✓✓ Each peak represents one base,
with colors corresponding to nucleotides (A = green, T = red, G = black, C = blue).
• What are the limitations of Sanger sequencing? -✓✓ Can only read ~500 nt reliably
and is not suitable for metagenomic samples.
• What is the first step in analyzing a chromatogram? -✓✓ Identify clear, non-
overlapping peaks.
• What is the purpose of using BLAST after sequencing? -✓✓ To determine taxonomy
by comparing the sequence to a nucleotide database.
, • What are the modifications required for DNA fragments before sequencing by
synthesis? -✓✓ Adapters, indexes (barcodes), and primer binding sites.
• What is the advantage of sequencing by synthesis (Illumina)? -✓✓ Very high accuracy
(~99%) and massively parallel sequencing.
• What is a disadvantage of nanopore sequencing? -✓✓ Lower accuracy (~92-97%)
compared to other methods.
• What is the purpose of using specific genes in PCR? -✓✓ To detect one species,
strain, or functional gene.
• What are universal genes used for in PCR? -✓✓ To amplify all members of a large
group of organisms.
• What is the role of conserved regions in universal genes? -✓✓ They are where
universal primers bind.
• What is the purpose of hypervariable regions in universal genes? -✓✓ Used to identify
organisms.
• What is the output format of Illumina sequencing? -✓✓ Two FASTQ files per sample
(forward + reverse reads).
• What is a key feature of nanopore sequencing technology? -✓✓ It allows real-time
sequencing without PCR.
• What is the significance of the E-value in BLAST results? -✓✓ It indicates the
statistical significance of the match.
• What does a high % identity in BLAST results indicate? -✓✓ It identifies the species
with high confidence.
• What is the purpose of trimming low-quality ends in chromatogram analysis? -✓✓ To
remove fuzzy, overlapping peaks for accurate sequence determination.
• What is the function of indexes (barcodes) in sequencing? -✓✓ To identify which
sample fragments came from during multiplexing.
• What is bridge amplification in Illumina sequencing? -✓✓ PCR amplification that
occurs directly on the flow cell to create clusters of identical DNA fragments.
• How does Nanodrop quantify DNA? -✓✓ It measures UV absorbance at 260 nm to
assess nucleic acids and reports total nucleic acid concentration.
