Molecular Biology Quiz
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Questions and Answers

What is the reason for the spontaneous depurination of purines in a cell each day?

  • The N-glycosyl linkage between deoxyribose and purines hydrolyzes. (correct)
  • The enzyme deaminase continually acts on purines.
  • Purines are inherently unstable within the cell.
  • It is a result of the high reactivity of purines.

Which feature of RNA contributes to its lower stability compared to DNA?

  • Higher molecular weight of RNA.
  • Absence of a hydroxyl group at the 2' position.
  • Presence of two phosphate groups.
  • Presence of a hydroxyl group at the 2' position. (correct)

What is one of the types of DNA damage that Nucleotide Excision Repair (NER) is capable of repairing?

  • Inter-strand crosslinks
  • Pyrimidine dimers (correct)
  • Base mismatches
  • Single-strand breaks

What is the consequence of 15 minutes of sun exposure on keratinocytes?

<p>Up to 10^5 lesions may arise. (D)</p> Signup and view all the answers

Which of the following agents is known to cause crosslinking in DNA?

<p>Cisplatin (B)</p> Signup and view all the answers

What is the primary purpose of the demultiplexer in the sequence processing workflow?

<p>To sort data by index into different samples. (C)</p> Signup and view all the answers

Which method of library preparation is characterized by the use of transposomes?

<p>Tagmentation (B)</p> Signup and view all the answers

What is a downside of the amplification method for library preparation?

<p>It is only effective if flanking regions are known. (A)</p> Signup and view all the answers

What is the role of the adapter sequences in library preparation?

<p>To facilitate the binding of transposases. (D)</p> Signup and view all the answers

Which of the following statements about sequencing by synthesis is TRUE?

<p>It includes both library preparation and sequencing steps. (B)</p> Signup and view all the answers

What is one of the major concerns with using CRISPR/Cas9 in gene editing related to its specificity?

<p>Sequence-dependent off-targeting (A)</p> Signup and view all the answers

What effect can a Cas9 mutant with lowered off-target activity have on gene editing?

<p>Decrease the risk of unintended edits (A)</p> Signup and view all the answers

What is one possible consequence of introducing a plasmid encoding Cas9 into the genome?

<p>Integration into the genome (B)</p> Signup and view all the answers

How can lowering the expression level of Cas9 affect its functionality?

<p>It reduces off-target cleavage risks. (C)</p> Signup and view all the answers

What should be considered when designing gRNA to minimize off-target effects?

<p>The location of mismatches relative to the PAM sequence (D)</p> Signup and view all the answers

What is a major challenge in metagenomics when characterizing microorganism communities?

<p>Representing the true composition of the community (A)</p> Signup and view all the answers

Which method allows for the analysis of specific regions in microbial communities?

<p>16S amplicon sequencing (A)</p> Signup and view all the answers

In cancer genome sequencing, what does SNP stand for?

<p>Single nucleotide polymorphism (B)</p> Signup and view all the answers

What is a primary benefit of using RNA sequencing (RNA-seq)?

<p>It provides insights into gene expression (A)</p> Signup and view all the answers

Which of the following best describes amplicon sequencing?

<p>A technique that targets specific DNA regions (D)</p> Signup and view all the answers

What is a significant outcome of analyzing 16S amplicon sequencing data in agriculture?

<p>Revealing shifts in microbial populations under environmental stress (C)</p> Signup and view all the answers

What role does the PAM sequence play in the function of cas9?

<p>It is recognized by cas9 to initiate DNA binding and cutting. (D)</p> Signup and view all the answers

Why is bulk experimentation used in genomic studies?

<p>To observe mixed outcomes from thousands or millions of cells (D)</p> Signup and view all the answers

Which statement accurately reflects the cutting capability of cas9?

<p>Cas9 cuts DNA in a precise manner three nucleotides upstream of PAM. (C)</p> Signup and view all the answers

What type of regions does PacBio sequencing aim to cover for maximum resolution?

<p>Variable and conserved regions (B)</p> Signup and view all the answers

What comes as a consequence of genome rearrangements in cancer genomes?

<p>Extreme genome aberrations and drug adaptation (A)</p> Signup and view all the answers

What is the significance of combining crRNA and tracrRNA into a single guide RNA (gRNA)?

<p>It simplifies the genome editing process. (A)</p> Signup and view all the answers

Which cas9 ortholog is predominantly discussed in the context of genome editing?

<p>Streptococcus pyogenes cas9 (C)</p> Signup and view all the answers

In what areas is metagenomics applied?

<p>Research fields like ecology and biotechnology (D)</p> Signup and view all the answers

What was a key outcome of the research conducted by Jinek and Charpentier in July 2012?

<p>They proposed a genome editing tool utilizing cas9, crRNA, and tracrRNA. (C)</p> Signup and view all the answers

What is the primary function of RNF8 in DNA damage signaling?

<p>Ubiquitination of histones (D)</p> Signup and view all the answers

During which phases of the cell cycle is homologous recombination (HR) possible?

<p>S phase and G2 (A)</p> Signup and view all the answers

Which of the following proteins are recruited to double-strand breaks (DSBs) during non-homologous end joining (NHEJ)?

<p>DNA-PK and Ku70/Ku80 (B)</p> Signup and view all the answers

What happens to the levels of gammaH2AX and BRCA1 after silencing RNF8?

<p>GammaH2AX levels remain the same while BRCA1 levels decrease (D)</p> Signup and view all the answers

Which proteins play a role in the short-range resection of DSBs during homologous recombination?

<p>PARP1 and MRN (C)</p> Signup and view all the answers

What is the role of RAD51 during homologous recombination?

<p>To displace RPA from ssDNA (B)</p> Signup and view all the answers

What are the consequences of Ku70/Ku80 binding to DSB ends?

<p>Activation of Artemis for end processing (D)</p> Signup and view all the answers

During which step of homologous recombination do polymerases extend the invading strand?

<p>Double Holliday junction formation (D)</p> Signup and view all the answers

Flashcards

Spontaneous Depurination

The process where a purine base (Adenine or Guanine) is removed from DNA due to the breaking of the N-glycosyl bond, which connects the purine base to the deoxyribose sugar. This happens naturally in the cell at a rate of 18,000 purines per cell per day.

Spontaneous Deamination

The process where an amine group (NH2) is removed from a base in DNA, often converting Cytosine to Uracil. This occurs spontaneously at a rate of 100-500 bases per cell per day.

RNA Stability Differences

RNA is less stable than DNA due to the presence of a hydroxyl group at the 2' position of its ribose sugar. This hydroxyl group can act as a nucleophile under alkaline conditions, hydrolysing the phosphodiester bond that connects nucleotides in the RNA chain, leading to degradation.

Nucleotide Excision Repair (NER)

A DNA repair mechanism that removes a wide range of damaged DNA segments, including bulky adducts and crosslinks, by excising a short stretch of nucleotides containing the damage and replacing it with the correct sequence.

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Bulky Adducts

These are molecules that bind to DNA and cause a significant distortion in its structure. Examples include N-hydroxyl-aminofluorene, Aflatoxin B1, and Benzo-a-pyrene, which often attach to guanine bases.

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What is a key step in determining if a protein is involved in DNA damage signaling?

Observe if the signaling step is disrupted when the protein is degraded, depleted, or knocked out.

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How do you determine the role of RNF8 in DNA damage signaling?

By silencing RNF8 and observing changes in the levels of gammaH2AX and BRCA1 through immunofluorescence.

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What are the two main pathways for repairing double-strand breaks (DSBs)?

Non-homologous end joining (NHEJ) and homologous recombination (HR).

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What is the key difference between NHEJ and HR in terms of cell cycle phases?

NHEJ can function throughout the cell cycle, while HR only works in S phase and G2.

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How does NHEJ repair DSBs?

Ku70/Ku80 bind to the ends, recruit DNA-PK, LIG4, XRCC4, and XLF. DNA-PK activates Artemis to process the ends, and LIG4/XRCC4/XLF ligate the ends.

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What is the role of PARP1 and MRN in HR?

They sense DSB ends and recruit CtIP-BRCA1 to initiate repair.

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Why is long-range resection important for HR?

It generates a long 3' ssDNA tail coated by RPA, which is essential for RAD51 filament formation and homologous DNA strand invasion.

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Briefly describe late stages of homologous recombination.

BRCA2 displaces RPA from ssDNA. RAD51 forms filaments on ssDNA, invades the homologous duplex, and forms a D-loop. DNA polymerases extend the invading strand, leading to double Holliday junction formation. Nucleases resolve Holliday junctions, generating crossovers.

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Tagmentation

A method for preparing DNA libraries by using transposomes, enzymes that cut DNA and insert adapter sequences. It's cheaper and faster than other methods.

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Transposomes

Enzymes called transposases, modified to be highly active and less specific. They cut DNA and insert adapter sequences during library preparation.

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Sequencing by Synthesis

A method used for DNA sequencing by adding fluorescently labeled nucleotides one at a time, capturing the signal, and then removing the nucleotide. This process is repeated to determine the DNA sequence.

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Library Preparation (Sequencing)

The process of preparing DNA fragments for sequencing. It involves breaking down DNA into smaller pieces, adding adapters for binding, and attaching index sequences for sample identification.

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Demultiplexing

The process of sorting sequenced reads based on their unique index sequences, allowing researchers to separate and analyze data from different samples.

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What does Cas9 recognize?

Cas9 recognizes a specific DNA sequence called the PAM sequence, typically NGG (where N can be any nucleotide).

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How does Cas9 cut DNA?

Cas9 makes a blunt cut three nucleotides upstream of the PAM sequence. This precise cut is crucial for targeted gene editing.

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Single guide RNA (gRNA)

gRNA is a single RNA molecule that combines the functions of crRNA and tracrRNA, simplifying the Cas9 system. It guides Cas9 to the target DNA sequence.

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What are Cas9 orthologs?

Cas9 orthologs are different versions of Cas9 found in various species. They may have different PAM sequences, sizes, and overall functionalities.

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Genome engineering with CRISPR-Cas9

CRISPR-Cas9 is a revolutionary technology used for precise genome editing. It allows scientists to modify DNA sequences in living organisms.

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Metagenomics

The study of the genetic material of entire microbial communities, analyzing DNA from environmental samples like soil or water.

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Amplicon Sequencing

A technique analyzing specific regions of DNA, often the 16S rRNA gene, to identify and classify microorganisms in a community.

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Shotgun Sequencing

A method to sequence the entire DNA of a sample, by fragmenting the DNA and sequencing all the pieces, then reassembling the fragments computationally.

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16S rRNA Gene

A conserved gene in bacteria used for phylogenetic analysis. Its variable regions allow for classification of different species.

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RNA Sequencing (RNA-seq)

A technique used to measure the abundance of mRNAs in a sample, providing information about gene expression and cellular processes.

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Bulk Sequencing

Analyzing genetic material from a cell or organism using one combined sample.

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Single Cell Sequencing

Analyzing genetic material from individual cells, allowing for the study of variations within a population.

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Cancer Genome Sequencing

Analyzing the DNA of tumor cells to identify genomic changes like mutations, deletions, and amplifications that contribute to cancer development.

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SNP Calling

Identifying single nucleotide polymorphisms (SNPs) in DNA sequences, which are variations in a single nucleotide at a specific position.

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Rearrangements in Cancer Genomes

Large-scale changes in the order of DNA segments in cancer cells, such as deletions, duplications, and translocations.

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Study Notes

DNA damage

  • DNA damage occurs constantly from both exogenous and endogenous factors.
  • Exogenous factors include exposure to mutagens like benzo-a-pyrene from smoke and UV light, which cause damage.
  • Radiation, like Chernobyl radiation, and chemotherapeutic agents also cause DNA damage.
  • Endogenous factors include hydrolysis (damaging through water), reactive oxygen species (ROS) and radicals, and replication/recombination errors.
  • The cell experiences approximately 30,000 endogenous DNA lesions per cell daily, mainly from hydrolysis.

DNA repair mechanisms

  • Base damage is repaired through base excision repair (BER).
  • Nucleotide damage from bulky adducts or bipyrimidine photoproducts is repaired through nucleotide excision repair (NER).
  • Interstrand crosslinks are repaired through NER or interstrand crosslink repair (ICLR).
  • Single strand breaks are repaired through single strand break repair (SSBR).
  • Double strand breaks are repaired through homologous recombination (HR) or non-homologous end joining (NHEJ).
  • Mismatch repair (MR) fixes mismatches during replication.

DNA repair defects

  • Mutations in DNA repair pathways can lead to a variety of disorders and increased susceptibility to cancer.
  • Xeroderma pigmentosum (XP) due to problems in NER, makes people vulnerable to ultraviolet (UV) light.

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Methods In Molecular Biology WS 2022 PDF

Description

Test your knowledge on key molecular biology concepts such as DNA damage, RNA stability, and library preparation techniques. This quiz covers various important topics including spontaneous depurination, Nucleotide Excision Repair, and sequencing methods.

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