#12: genome annotation

What was a major challenge in assembling the human genome from short DNA fragments?
A) Lack of sequencing technology
B) Presence of multiple start codons
C) Presence of repetitive DNA sequences
D) DNA degradation during sequencing

Presence of repetitive DNA sequences

Paired-end sequencing in BAC clones was particularly useful because:
A) It extended the read length of each sequence to over 300,000 bp
B) It identified repetitive sequences with high accuracy
C) It provided both sequence and spatial information about DNA fragments
D) It allowed for faster replication of BACs in bacteria

It provided both sequence and spatial information about DNA fragments

What is the approximate size of the DNA inserts typically carried by BACs used in genome sequencing?
A) 1,000–2,000 bp
B) 10,000–20,000 bp
C) 50,000–100,000 bp
D) 200,000–300,000 bp

200,000–300,000 bp

Which method allowed researchers to determine how to connect small DNA sequences into larger ones?
A) Southern blotting
B) Paired-end sequencing of BAC clones
C) CRISPR-Cas9 mapping
D) Polymerase Chain Reaction (PCR)

Paired-end sequencing of BAC clones

The challenge in genome assembly due to ____________ sequences arises because identical or nearly identical DNA segments occur in multiple locations.

repetitive

BACs, or ____________, are engineered plasmids that can carry large DNA fragments and are used to help assemble genomes.

bacterial artificial chromosomes

T/F: The use of BAC clones helped mitigate the problem of repetitive DNA by offering contextual placement of sequence fragments.

true

T/F: Repetitive sequences made genome assembly easier by allowing fragments to match multiple regions.

false

T/F: Each BAC clone contains a large DNA fragment, and sequencing both ends helps in determining the correct order of smaller sequences.

true

Why is matching RNA sequences back to the genome useful in identifying genes?
A) RNA is always longer than DNA
B) Transcribed regions indicate likely gene locations
C) RNA contains unique methylation patterns
D) RNA can only bind to protein-coding regions

Transcribed regions indicate likely gene locations

Which of the following is not an exception to the central dogma?
A) tRNA
B) rRNA
C) mRNA
D) Long non-coding RNA

mRNA

he process of using RNA transcripts to locate genes in the genome is based on the idea that if a DNA sequence is ____________, it is likely part of a gene.

transcribed

T/F: Matching mRNA sequences to DNA can help scientists identify coding regions of the genome.

true

Why can DNA be read in six different reading frames?
A) There are six types of stop codons
B) Each strand has three possible reading frames
C) DNA has six types of nucleotides
D) Each codon can be read backwards and forwards

Each strand has three possible reading frames

hat is the significance of a long open reading frame (ORF) in DNA sequence analysis?
A) It indicates a regulatory region
B) It suggests the presence of a gene
C) It marks the end of a chromosome
D) It always codes for tRNA

It suggests the presence of a gene

On average, how frequently should a stop codon appear in randomly distributed nucleotide triplets?
A) Every 3 triplets
B) Every 21 triplets
C) Every 64 triplets
D) Every 100 triplets

Every 21 triplets

Which of the following best defines an open reading frame (ORF)?
A) A sequence of DNA that includes only stop codons
B) A continuous sequence of codons between two genes
C) A series of codons without a stop codon starting from a specific point
D) A promoter sequence that signals gene expression

A series of codons without a stop codon starting from a specific point

Because each DNA strand can be read in three directions, and there are two strands, DNA has ____________ possible reading frames.

6

If a stretch of DNA contains a long sequence without any stop codons, it is likely to represent a ____________.

gene

T/F: Long open reading frames are typically found in non-coding regions of the genome.

false

Why are some DNA sequences highly conserved across species?
A) They mutate faster than other sequences
B) They are not transcribed or translated
C) They code for essential functions and are maintained by natural selection
D) They are found only in non-coding regions

They code for essential functions and are maintained by natural selection

What does DNA homology between two species suggest?
A) They evolved independently
B) They have identical mutation rates
C) They likely share a common ancestor
D) Their DNA is not functional

They likely share a common ancestor

Which of the following best describes a "conserved sequence"?
A) A sequence repeated in only one species
B) A DNA segment present only in recent species
C) A DNA sequence that remains similar across species due to evolutionary importance
D) A gene only found in bacteria

A DNA sequence that remains similar across species due to evolutionary importance

What happens to harmful mutations over time through natural selection?
A) They are conserved
B) They are amplified in the population
C) They are typically eliminated
D) They become part of conserved sequences

They are typically eliminated

Mutations that are beneficial to an organism are typically ____________ through natural selection.

retained

The similarity in DNA sequences between different species due to shared ancestry is known as DNA ____________.

homology

T/F: All DNA sequences evolve at the same rate over time.

false

T/F: Homologous DNA sequences are similar due to random mutation.

false

What role does reverse transcriptase play in retroviral infection?
A) It copies host DNA into viral RNA
B) It converts host proteins into viral enzymes
C) It converts viral RNA into DNA
D) It integrates viral DNA into the nucleus

It converts viral RNA into DNA

Why is reverse transcriptase widely used in molecular genetics?
A) It improves RNA stability
B) It enables the synthesis of complementary DNA (cDNA) from RNA
C) It directly sequences DNA
D) It can remove introns from genomic DNA

It enables the synthesis of complementary DNA (cDNA) from RN

Which of the following is true about retroviruses?
A) They store their genetic information in DNA
B) They replicate entirely outside the host cell
C) They use RNA as their genetic material and integrate DNA into the host genome
D) They use double-stranded DNA as their primary infectious form

They use RNA as their genetic material and integrate DNA into the host genome

After reverse transcription, the viral DNA is:
A) Degraded by the host cell
B) Used only to make RNA
C) Integrated into the host genome
D) Sent back outside the cell

Integrated into the host genome

Retroviruses carry their genetic information in the form of ____________.

RNA

The enzyme that converts RNA into DNA in retroviruses is called ____________.

reverse transcriptase

T/F: Reverse transcription is essential for retroviruses to insert their genetic material into host DNA.

true

T/F: The genetic material of retroviruses is initially in the form of DNA.

false

The process of synthesizing DNA from an RNA template is known as ____________.

reverse transcription

What feature allows mRNA to be selectively isolated from a mixture of RNAs?
A) Its short length
B) Its secondary structure
C) Its poly-A tail
D) Its circular shape

Its poly-A tail

Why do scientists use magnetic beads coated with oligo-dT in mRNA purification?
A) To degrade unwanted RNA
B) To attach to the 5' cap of mRNA
C) To base pair with the poly-A tail of mRNA
D) To hybridize with rRNA sequences

) To base pair with the poly-A tail of mRNA

What happens when a magnet is placed next to the tube containing oligo-dT beads and RNA?
A) All RNA types move toward the magnet
B) mRNA remains in solution
C) Beads with mRNA attached are pulled to one side
D) RNA degrades in the magnetic field

Beads with mRNA attached are pulled to one side

What type of molecule results from the reverse transcription of mRNA?
A) RNA-RNA duplex
B) Protein-RNA complex
C) DNA-RNA hybrid
D) Double-stranded DNA

DNA-RNA hybrid

The poly-A tail on mRNA is composed entirely of the nucleotide ____________.

adenine

Oligo-dT beads are coated with short DNA strands made entirely of the nucleotide

thymine

mRNA can be purified from total RNA because it binds to oligo-dT beads via ____________ base pairing.

adenine-thymine

T/F: All types of RNA have a poly-A tail and bind to oligo-dT beads.

false

T/F: The poly-A tail is unique to eukaryotic mRNA and is used for selective purification.

true

T/F: Magnetic separation allows scientists to isolate mRNA from other RNA types in a cell extract.

true

What is the function of RNase in the cDNA synthesis process?
A) It synthesizes the second strand of DNA
B) It removes the mRNA template after the first DNA strand is made
C) It converts mRNA to DNA
D) It creates the 3'OH end

It removes the mRNA template after the first DNA strand is made

What structure forms at the end of the single-stranded DNA that allows DNA polymerase to begin synthesis of the second strand?
A) Poly-A tail
B) Promoter region
C) Hairpin loop
D) Spliceosome

Hairpin loop

Why is the 3′OH group important in cDNA synthesis?
A) It blocks DNA synthesis
B) It signals RNase to degrade RNA
C) It serves as the starting point for DNA polymerase
D) It connects exons during splicing

It serves as the starting point for DNA polymerase

What is the role of a nuclease during cDNA synthesis?
A) It adds nucleotides to growing DNA strands
B) It removes introns from mRNA
C) It cuts the hairpin loop to form double-stranded cDNA
D) It transcribes mRNA

It cuts the hairpin loop to form double-stranded cDNA

The enzyme that digests the RNA strand after the first DNA strand is synthesized is called ____________.

RNase

After the first strand of DNA is made, the molecule folds to form a ____________, which provides a free 3′OH group.

hairpin loop

DNA polymerase synthesizes the second strand of DNA using the ____________ DNA strand as a template.

first

A cDNA molecule is a DNA copy made from ____________ using reverse transcriptase.

mRNA

T/F: RNase digests the DNA strand during cDNA synthesis.

false

T/F: DNA polymerase cannot begin synthesis without a free 3′OH group.

true

T/F: A single-stranded cDNA can be converted into double-stranded cDNA by DNA polymerase.

true

What is a major drawback of traditional gene cloning using plasmids?
A) It lacks specificity
B) It generates too many sequencing errors
C) It is time-consuming and costly
D) It produces only RNA, not DNA

It is time-consuming and costly

How does Next Generation Sequencing (NGS) address the limitations of traditional cloning methods?
A) It amplifies proteins directly
B) It replaces RNA with DNA in cells
C) It enables high-throughput and automated sequencing
D) It eliminates the need for computers in sequence analysis

It enables high-throughput and automated sequencing

NGS, or ____________, allows millions of DNA fragments to be sequenced in parallel.

next generation sequencing

What is the primary purpose of the Polymerase Chain Reaction (PCR)?
A) To transcribe RNA from DNA
B) To insert genes into plasmids
C) To amplify specific DNA sequences
D) To sequence entire genomes

To amplify specific DNA sequences

What role do primers play in the PCR process?
A) They stabilize the DNA strands
B) They act as templates for transcription
C) They define the start and end points of the DNA region to be amplified
D) They break down DNA into smaller pieces

They define the start and end points of the DNA region to be amplified

Why is DNA polymerase essential in PCR?
A) It transcribes mRNA
B) It binds the DNA strands together
C) It synthesizes new DNA strands using primers and template DNA
D) It degrades RNA contaminants

It synthesizes new DNA strands using primers and template DNA

What is the typical size limit for a DNA fragment that can be efficiently amplified using standard PCR?
A) 100 base pairs
B) 1 kilobase
C) 10 kilobases
D) 100 kilobases

10 kilobases

PCR allows scientists to make ____________ of copies of a specific DNA fragment.

thousands to millions

What happens during the denaturing step of PCR?
A) DNA polymerase adds nucleotides to the strand
B) Primers bind to template DNA
C) The DNA double helix is separated into single strands
D) RNA is converted into DNA

The DNA double helix is separated into single strands

During the annealing step of PCR, the temperature is lowered primarily to:
A) Inactivate DNA polymerase
B) Allow primers to bind to complementary DNA sequences
C) Cause DNA strands to re-anneal randomly
D) Separate protein contaminants

Allow primers to bind to complementary DNA sequences

Which of the following best describes the role of the 3′OH group during PCR?
A) It degrades the DNA strand
B) It prevents primer binding
C) It serves as the starting point for DNA polymerase
D) It signals the end of DNA replication

It serves as the starting point for DNA polymerase

What happens in the extension step of PCR?
A) DNA strands are separated
B) DNA polymerase synthesizes a new strand using the primer
C) RNA is spliced into the DNA
D) Primer dimers are removed

DNA polymerase synthesizes a new strand using the primer

he PCR step that separates double-stranded DNA is called ____________.

denaturing

During the annealing step, primers bind to the ____________ strand by base pairing.

template

DNA polymerase extends the new DNA strand from the primer’s free ____________ group.

3’OH

The extension step in PCR occurs after primers have ____________ to the DNA template.

annealed (bound)

T/F: Denaturing requires a high temperature to break hydrogen bonds between DNA strands.

true (94-94 degrees celsius)

T/F: Annealing temperatures must be higher than denaturing temperatures to prevent non-specific binding.

false (40-65 degrees celsius)

T/F: The three PCR steps—denaturing, annealing, and extension—repeat in cycles to amplify DNA.

true

How do primers influence the PCR amplification process?
A) They determine which DNA fragment is copied
B) They increase the temperature needed for denaturation
C) They degrade non-target DNA sequences
D) They directly synthesize new DNA strands

They determine which DNA fragment is copied

Why do PCR reactions usually include 35-40 cycles?
A) To degrade original DNA templates
B) To produce millions of copies of the target DNA
C) To allow primers to degrade gradually
D) To prevent DNA polymerase activity

To produce millions of copies of the target DNA

After several PCR cycles, what role do newly synthesized DNA strands play?
A) They are discarded as waste
B) They serve as templates for further amplification
C) They inhibit primer binding
D) They convert back to RNA

They serve as templates for further amplification

Which statement best describes PCR amplification?
A) Random DNA regions are amplified each cycle
B) Only one copy of DNA is made per cycle
C) The exact same DNA fragment is copied repeatedly
D) DNA synthesis occurs without primers

The exact same DNA fragment is copied repeatedly

The number of PCR cycles typically ranges between __________ and __________.

35;40

T/F: PCR cycles duplicate the amount of target DNA exponentially.

true

What is a main limitation of Sanger sequencing?
A) It produces highly inaccurate reads
B) It sequences millions of DNA fragments simultaneously
C) It sequences only one DNA fragment at a time, making it slow for large-scale projects
D) It cannot sequence DNA longer than 100 base pairs

It sequences only one DNA fragment at a time, making it slow for large-scale projects

Which of the following describes high-throughput sequencing?
A) Sequencing one cloned DNA piece at a time
B) Simultaneous sequencing of millions of random DNA fragments
C) Uses chain-terminating nucleotides only
D) Produces reads only 50 base pairs long

Simultaneous sequencing of millions of random DNA fragments

How are longer sequences reconstructed in high-throughput sequencing?
A) By reading single strands only
B) By sequencing plasmids one by one
C) By assembling shorter overlapping reads using bioinformatics
D) By sequencing RNA instead of DNA

By assembling shorter overlapping reads using bioinformatics

What is the typical read length for Sanger sequencing?
A) 50–100 base pairs
B) 600–700 base pairs
C) Over 10,000 base pairs
D) Only a few base pairs

600–700 base pairs

Sanger sequencing is also known as ____________ sequencing.

chain termination

In high-throughput sequencing, shorter DNA reads are combined by finding ____________ in their sequences.

overlaps

T/F: High-throughput sequencing is generally faster and cheaper than Sanger sequencing for large-scale projects.

true

Why is DNA fragmented during library preparation for high-throughput sequencing?
A) To make DNA easier to amplify and sequence
B) To remove non-coding regions
C) To convert DNA into RNA
D) To sequence only protein-coding genes

To make DNA easier to amplify and sequence

What is the primary function of adaptor sequences ligated to DNA fragments in sequencing libraries?
A) They protect DNA from degradation
B) They serve as binding sites for sequencing primers and PCR amplification
C) They cleave the DNA into smaller pieces
D) They translate DNA into proteins

They serve as binding sites for sequencing primers and PCR amplification

Which of the following is NOT a purpose of adaptor sequences in high-throughput sequencing?
A) Hybridization during sequencing
B) Acting as primers for PCR
C) Serving as identifiers for bioinformatic analysis
D) Encoding the target gene sequence

Encoding the target gene sequence

Library preparation involves attaching adaptors to:
A) Only one end of the DNA fragment
B) Both ends of the DNA fragment
C) Only coding regions of DNA
D) RNA molecules

Both ends of the DNA fragment

In library preparation, DNA is first ____________ into smaller fragments.

fragmented

Adaptors ligated to DNA fragments are important for ____________ and sequencing reactions.

PCR

Adaptors can serve as ____________ during bioinformatic analysis to identify specific sequences.

identifiers

DNA fragments used in high-throughput sequencing are usually ____________ stranded.

double

What is the main function of the flowcell in sequencing by synthesis?
A) To fragment DNA into smaller pieces
B) To hold and anchor DNA fragments during sequencing
C) To synthesize adaptor sequences
D) To convert RNA into DNA

To hold and anchor DNA fragments during sequencing

How do DNA fragments attach to the flowcell surface?
A) By covalent bonding to glass
B) By hybridization between adaptor sequences on DNA and complementary sequences on the flowcell
C) By random adsorption to the slide
D) By enzymatic ligation to the surface

By hybridization between adaptor sequences on DNA and complementary sequences on the flowcell