Podcast
Questions and Answers
What is the primary role of CTCF in chromatin looping?
What is the primary role of CTCF in chromatin looping?
How does WAPL affect gene expression in the context of chromatin structure?
How does WAPL affect gene expression in the context of chromatin structure?
What consequence can result from mutations in WAPL related to chromatin loops?
What consequence can result from mutations in WAPL related to chromatin loops?
Which structural element is critical for the action of cohesin in gene regulation?
Which structural element is critical for the action of cohesin in gene regulation?
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What characteristic is essential for primer design to ensure specificity in PCR?
What characteristic is essential for primer design to ensure specificity in PCR?
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What is the purpose of cloning in DNA technology?
What is the purpose of cloning in DNA technology?
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Which type of restriction enzyme cuts within recognition sequences?
Which type of restriction enzyme cuts within recognition sequences?
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EcoRI creates ____ ends at 5'…GAATTC…3'.
EcoRI creates ____ ends at 5'…GAATTC…3'.
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What is the role of DNA ligase in molecular cloning?
What is the role of DNA ligase in molecular cloning?
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What is blue-white screening used for in cloning?
What is blue-white screening used for in cloning?
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What does TA cloning utilize to ligate PCR products?
What does TA cloning utilize to ligate PCR products?
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Lambda phage is suitable for inserting up to 25 kb of DNA.
Lambda phage is suitable for inserting up to 25 kb of DNA.
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How is mRNA isolated from other RNAs?
How is mRNA isolated from other RNAs?
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What is the purpose of sequencing cDNA clones?
What is the purpose of sequencing cDNA clones?
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Antibodies are composed of two identical heavy chains and two identical ____ chains.
Antibodies are composed of two identical heavy chains and two identical ____ chains.
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Which of the following is an example of a plasmid vector?
Which of the following is an example of a plasmid vector?
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Study Notes
Cohesin and CTCF in Genome Looping
- Cohesin and CTCF work together to form chromatin loops, which are essential for regulating gene expression.
- CTCF acts as an anchor, controlling cohesin's movement on DNA.
- Interactions between cohesin and CTCF stabilize these chromatin loops.
WAPL's Role in Chromatin Structure
- WAPL regulates the release of cohesin from chromatin, which influences the accessibility of genes.
- Mutations in WAPL disrupt chromatin loops, affecting gene expression.
- These disruptions may contribute to diseases like cancer.
V(D)J Recombination and Cancer
- V(D)J recombination utilizes loop extrusion to bring distant gene segments together, contributing to immune diversity.
- Disruptions in CTCF binding or cohesin control during this process are linked to cancer development.
Structural Insights into Cohesin and WAPL Interactions
- Interactions between SA2 and SCC1 in the cohesin complex impact the function of WAPL.
- Specific mutations within the cohesin complex, such as SCC1 P355L, affect WAPL function, influencing cohesin release.
- These mutations are associated with cancers such as acute lymphoblastic leukemia and adenocarcinoma.
Centromeric Cohesion Protection
- SGO1 safeguards centromeric cohesin from WAPL-mediated release, ensuring chromosome stability during cell division.
- Mutations in SGO1 disrupt centromeric cohesion, leading to chromosome segregation errors and genetic instability.
Primer Design Basics
- Primers should be approximately 20 bases long with a 45-55% GC content.
- Primer design should avoid self-dimers and hairpins.
- The final base of a primer should preferably be G or C for stability.
Advanced Primer Considerations
- Allele-specific primers are used to detect single-nucleotide polymorphisms (SNPs).
- Computer-aided tools like Primer3 and in silico PCR assist in ensuring primer specificity.
PCR Fidelity and Errors
- Taq polymerase lacks proofreading capabilities and is prone to errors.
- Alternative polymerases like Pfu are used for higher fidelity PCR applications.
- Some Taq polymerases add an A overhang, which is beneficial for TA cloning.
DNA Cloning in E.coli
- Purpose of Cloning: Creating recombinant DNA molecules, identifying genes, producing specific proteins.
- Key Steps in Cloning: Ligation of foreign DNA into a plasmid vector, transformation into E.coli, selection of recombinant clones.
Restriction Enzymes
- Function: Cut DNA at specific sequences (palindromic sites).
- Type II Restriction Enzymes: Cut within recognition sequences, leaving blunt or sticky ends.
- Isoschizomers: Enzymes recognizing the same sequence but cutting differently.
- Example: EcoRI creates sticky ends at 5'…GAATTC…3'.
Ligation and Cloning
- Ligation: DNA ligase joins DNA fragments using ATP to seal the phosphodiester bond between 5’ and 3’ ends.
- Ligation Conditions: Low DNA concentration promotes intramolecular ligation (circle formation), while high concentration promotes intermolecular ligation (recombinants).
Vector Considerations
-
Plasmid Vectors:
- pBR322: Contains ampicillin and tetracycline resistance.
- pUC19: Contains lacZ for blue-white screening (inactivation leads to white colonies for recombinants).
-
Other Vectors:
- Lambda phage: 5-25 kb insert.
- Cosmid: 35-45 kb insert.
- BAC: Up to 300 kb insert.
Transformation Methods
- Chemical Transformation: Uses divalent ions (e.g., Ca²⁺) and heat shock to introduce DNA into cells.
- Electroporation: Uses an electric pulse to create membrane pores, allowing DNA entry.
Screening for Recombinants
- Blue-White Screening: Recombinants disrupt lacZ, producing white colonies on X-Gal medium.
- Library Screening: Hybridization, PCR, or protein expression assays identify desired clones.
PCR Cloning
- PCR Primers: Can include restriction sites for easier cloning.
- TA Cloning: Uses vectors with T residues to ligate PCR products with A overhangs (e.g., Promega TA Cloning vector).
Advanced Methods
- Gibson Assembly: Utilizes exonucleases and DNA polymerase to assemble DNA fragments without restriction sites.
- Topo Cloning: Vectors have topoisomerase to ligate blunt-end PCR products efficiently.
Practical Considerations
- Host Strain: E.coli strains used in cloning are often engineered to improve plasmid stability (e.g., recA⁻ to reduce recombination).
- Plasmid Purification: Methods include alkaline lysis, anion-exchange resins, and CsCl-EtBr gradient centrifugation for high-quality plasmid extraction.
cDNA Libraries
- Human Genome: Contains ~21,000 protein-coding genes within ~3x10⁹ bp.
- mRNA: Contains protein-coding sequences (~1% of the genome), derived from hnRNA through splicing.
- hnRNA & mRNA: Both have a 5' cap and a 3' poly-A tail for stability.
Polyadenylation and mRNA Isolation
- mRNA: ~1-2% of total RNA, separated using affinity chromatography due to its poly(A) tail.
- Poly(A) Tails: ~200 nucleotides, stabilize mRNA and are added post-transcriptionally.
cDNA Production
- Reverse Transcriptase: Converts mRNA into a complementary DNA strand.
- Second Strand Synthesis: Various methods, like Gubler and Hoffman's, use RNase H and DNA polymerase I.
- Ligation into Plasmid Vectors: Can be inefficient, adding adapters improves the process.
Library Screening
- cDNA Libraries: Contain all mRNA sequences from the sample; rare mRNAs are also rare in the library.
- Screening Methods: In situ colony screening and hybridization probes are used to identify the desired cDNA clone.
Screening Techniques
- Oligonucleotide Probes: Designed from protein sequences, suitable for cDNA but not genomic libraries.
- Antibody Screening: Detects expressed cDNA in expression vectors by binding to specific proteins.
Confirmation and Conclusion
- Sequencing: Confirms the correctness of the cDNA clone, typically using the Sanger method.
- cDNA: Lacks non-coding regions, improved methods focus on efficiency and yield in cDNA cloning.
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Description
Explore the crucial roles of cohesin and CTCF in forming chromatin loops for gene regulation. Learn about the impact of WAPL on chromatin structure and how disruptions can lead to diseases like cancer. This quiz delves into the complexities of V(D)J recombination and the structural interactions in the cohesin complex.
