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?

Random mutations, primarily deletions Signup and view all the answers

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

At least 95% homology is needed over a length of at least 1000 bp. Signup and view all the answers

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

S phase and G2 Signup and view all the answers

Which statement about CRISPR-Cas9 technology is true?

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

What are the potential uses of HR in genome editing?

To facilitate targeted insertion of DNA molecules and gene knock-ins. Signup and view all the answers

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

They are among the most deleterious DNA lesions. Signup and view all the answers

Which process contributes to mutagenesis during DNA repair?

NHEJ Signup and view all the answers

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

The presence of a protospacer adjacent motif (PAM). Signup and view all the answers

How does CRISPR-Cas9 technology facilitate genome editing?

By creating targeted double-strand breaks Signup and view all the answers

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

Small deletions that can lead to frameshift mutations. Signup and view all the answers

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

NHEJ is error-prone, while HR is error-free. Signup and view all the answers

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

It becomes more prevalent as S phase progresses. Signup and view all the answers

What do large deletions during genome editing often result in?

Gene knockouts or complete loss-of-function. Signup and view all the answers

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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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.