MIC 115 Lecture 14: Recombinant DNA Cloning
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Questions and Answers

What is the primary characteristic of homologous recombination (HR) in DNA repair?

  • Operates primarily in somatic cells
  • Uses sister chromatids for repair (correct)
  • Requires extensive trimming of DNA ends
  • Error-prone with random repair

Which type of DNA repair mechanism is predominantly used in somatic cells?

  • NHEJ (correct)
  • Homologous recombination
  • Error-free restoration
  • DNA repair by base excision

What role does homologous recombination (HR) play in meiotic cells?

  • It contributes to genetic diversity through crossovers. (correct)
  • It ensures the chromosomes remain identical after meiosis.
  • It only occurs in diploid cells during the G1 phase.
  • It prevents genetic diversity by limiting allele combinations.

What outcome does non-homologous end joining (NHEJ) typically lead to?

<p>Random mutations, primarily deletions (A)</p> Signup and view all the answers

What is the requirement for homologous recombination to occur between sequences on different chromosomes?

<p>At least 95% homology is needed over a length of at least 1000 bp. (D)</p> Signup and view all the answers

During which cell cycle phases does homologous recombination (HR) occur?

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

Which statement about CRISPR-Cas9 technology is true?

<p>It can induce double-strand breaks (DSBs) at specific genome locations. (A)</p> Signup and view all the answers

What are the potential uses of HR in genome editing?

<p>To facilitate targeted insertion of DNA molecules and gene knock-ins. (A)</p> Signup and view all the answers

Which of the following statements best describes the nature of double-strand breaks (DSBs) in DNA?

<p>They are among the most deleterious DNA lesions. (A)</p> Signup and view all the answers

Which process contributes to mutagenesis during DNA repair?

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

What aspect of CRISPR-Cas9 technology is critical for its function?

<p>The presence of a protospacer adjacent motif (PAM). (D)</p> Signup and view all the answers

How does CRISPR-Cas9 technology facilitate genome editing?

<p>By creating targeted double-strand breaks (A)</p> Signup and view all the answers

What is a potential outcome of using non-homologous end joining (NHEJ) in gene editing?

<p>Small deletions that can lead to frameshift mutations. (B)</p> Signup and view all the answers

What is a key difference between HR and NHEJ in terms of error rates?

<p>NHEJ is error-prone, while HR is error-free. (A)</p> Signup and view all the answers

Which of the following best describes homologous recombination during S phase?

<p>It becomes more prevalent as S phase progresses. (C)</p> Signup and view all the answers

What do large deletions during genome editing often result in?

<p>Gene knockouts or complete loss-of-function. (C)</p> Signup and view all the answers

Flashcards

DSB repair

Two main ways cells fix broken DNA strands (double-strand breaks).

Homologous Recombination (HR)

Accurate DNA repair using a template (similar DNA sequence).

Non-homologous End Joining (NHEJ)

Quick DNA repair method, potentially inaccurate, can cause small deletions.

NHEJ mutations

NHEJ can result in small deletions in the DNA.

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HR in dividing cells

Error-free DNA repair in cells during cell division using sister chromatids.

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Sister chromatids

Identical copies of a chromosome, created during DNA replication.

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Genome editing

Changing DNA in cells for various purposes, like treating diseases.

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CRISPR-Cas9

A powerful tool for genome editing, like a molecular scissor.

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Early vs. Late Replication

During DNA replication (S phase), different regions of the genome replicate at different times. Some regions replicate early, others later.

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Homologous Recombination (HR) in Meiosis

In meiosis, homologous chromosomes exchange genetic material through crossing over. This shuffling of maternal and paternal alleles contributes to genetic diversity.

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HR: Beyond Homologous Chromosomes

Homologous recombination can also occur between similar DNA sequences located on different chromosomes, even if they're not homologous chromosomes.

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Ectopic Recombination

HR between non-homologous chromosomes can lead to 'ectopic crossing over,' where segments of different chromosomes exchange places.

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HR for Gene Manipulation

Homologous recombination allows for targeted changes in DNA sequences by using exogenous DNA as a template. This is the foundation for genome editing.

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CRISPR-Cas9 System

Consists of a bacterial protein (Cas9) that cuts DNA and a guide RNA that directs Cas9 to a specific target sequence.

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CRISPR-Cas9: PAM

A specific DNA sequence (PAM) must be present near the target site for the CRISPR-Cas9 system to recognize and cut the DNA.

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Genome Editing by HR

CRISPR-Cas9-induced DNA breaks can be repaired by HR, allowing for precise insertion of DNA molecules or gene modifications.

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Study Notes

MIC115 Recombinant DNA Cloning - Lecture 14

  • Genome Editing: Two major DNA double-strand break (DSB) repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ)
  • NHEJ: Error-prone, random repair, predominant in somatic cells, responsible for small deletions.
  • HR: Error-free, template-based repair, uses sister chromatids, predominant in germ cells.
  • Genome Editing by NHEJ: Small deletions often occur due to trimming during the repair process
  • Genome Editing by HR: HR can be used for gene targeting, including knock-in, knockout, and large deletions, utilizing exogenous DNA.
  • CRISPR-Cas9: A technology that induces DSBs at specific sites in the genome, enhancing the efficiency of gene targeting.
  • CRISPR-Cas9 Mechanism: Uses a Cas9 nuclease (bacterial protein) and a single guide RNA (sgRNA) to create sequence-specific DSBs.
  • Nobel Prize (2020): Awarded to Emmanuelle Charpentier and Jennifer A. Doudna for their work on CRISPR-Cas9.
  • Homologous Recombination (HR) in Meiotic Cells: Uses homologous chromosomes to create genetic diversity via crossovers, shuffling maternal and paternal alleles in a specific chromosome.
  • HR in Mitotic Cells: Utilizes sister chromatids for error-free repair
  • HR in Mammalian Cells: Can introduce exogenous DNA into a gene, altering the gene. Flanking sequences at least 20 base pairs long are essential for this process
  • HR in Budding Yeast: Recombinant DNA with 20-base-pair flanking sequences can be easily exchanged for a homologous chromosomal site, making genome editing highly efficient.
  • Drug Resistance: Methods to identify cells in which DNA is properly repaired including markers such as G418.

CRISPR/Cas9 - Specifics

  • PAM (Protospacer Adjacent Motif): Sequence (NGG) needed for CRISPR-Cas9 to recognize and cut DNA
  • Targeted Insertion: CRISPR-Cas9 can be used to target insertion of DNA molecules into model organisms, such as yeast and mammalian cells.

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MIC115 Recombinant DNA Cloning Lecture 14 Notes PDF

Description

Explore the mechanisms of genome editing in this lecture on recombinant DNA cloning. Learn about two major DNA double-strand break repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ), and the role of CRISPR-Cas9 in enhancing gene targeting. This comprehensive overview includes discussions on error-prone and error-free repair processes and highlights the Nobel Prize-winning work in this field.

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