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Questions and Answers
What is the main purpose of homologous recombination in gene cloning?
What is the main purpose of homologous recombination in gene cloning?
Which step in DNA extraction is primarily responsible for breaking down the cell membranes?
Which step in DNA extraction is primarily responsible for breaking down the cell membranes?
How does the pUC plasmid facilitate the visual selection of colonies with donor DNA inserts?
How does the pUC plasmid facilitate the visual selection of colonies with donor DNA inserts?
What role do Na+ ions play in the DNA precipitation step during extraction?
What role do Na+ ions play in the DNA precipitation step during extraction?
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What is the main principle behind the purification of plasmid DNA from bacterial DNA?
What is the main principle behind the purification of plasmid DNA from bacterial DNA?
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What is a primary characteristic of plasmids as vectors?
What is a primary characteristic of plasmids as vectors?
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Which component is essential for a plasmid vector to function effectively?
Which component is essential for a plasmid vector to function effectively?
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What is the role of DNA ligase in gene cloning?
What is the role of DNA ligase in gene cloning?
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Why is integration of target genes into the host chromosome preferable in certain scenarios?
Why is integration of target genes into the host chromosome preferable in certain scenarios?
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What characteristic distinguishes type II endonucleases from other restriction enzymes?
What characteristic distinguishes type II endonucleases from other restriction enzymes?
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Study Notes
Chromosome integration
- Integration of the target genes into the host chromosome.
- A preferable strategy to overcome the drawbacks of plasmid-based overexpression.
- Useful for creating plasmid-free stable mutants.
- Possible in bacteria, but usually necessary in eukaryotes.
Transposon-mediated gene transposition
- Site-specific recombination occurs between pairs of defined sequences (target sites).
- Process mediated by a specific recombinase that can be expressed via a helper plasmid.
Homologous recombination
- A single crossover between a targeting gene and a homologous DNA fragment on a chromosome.
- The whole plasmid sequence is integrated.
DNA extraction
- DNA extraction (isolation) is a method to purify DNA by using physical and/or chemical methods.
- Separates DNA from cell membranes, proteins, and other cellular components.
- Aims to take only DNA from the whole cell extract.
- Basic steps:
- Lysis: the cell and the nucleus are broken open to release the DNA (mechanical disruption or chemical lysis with detergents).
- Precipitation: separation of DNA from the cellular debris.
- Purification: rinsed with alcohol to remove remaining unwanted material.
- Elution: with either the elution buffer or with water.
Extraction of plasmids
- Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis.
- Neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized.
- Chromosomal DNA and proteins are removed by centrifugation.
Cut and paste DNA: Plasmids
- Plasmids are circular, self-replicating, double-stranded DNA (dsDNA) molecules that are separate from a cell's chromosomal DNA.
- Plasmids have been engineered to optimize their use as vectors:
- Restriction sites.
- Marker genes for selection and/or screening.
- Origin of replication.
- Plasmid vectors replicate along with their host cells (low, medium or high copy number).
- A DNA fragment of a few base pairs up to ≈20 kb can be inserted into a plasmid vector.
- Like the host-cell chromosomal DNA, pDNA is duplicated before every cell division.
Cut and paste DNA: Eukaryotic expression vectors
- Eukaryotes: viruses
- Restriction sites.
- Virus genes.
- Terminal repeats.
Cut and paste DNA: Restriction
- Restriction sites are specific 4- to 8-bp sequences that are recognized by restriction endonucleases (restrictases).
- Many restriction sites are short inverted repeat sequences.
- Restriction enzyme type 2 cut DNA:
- Highly specific (type II endonucleases).
- Leaves blunt or sticky ends.
Cut and paste DNA: Ligation
- DNA ligase catalyzes formation of 3’ ? 5′ covalent bonds between the short fragments.
- Restriction fragments are covalently ligated together:
- Restriction fragments with complementary “sticky ends” are ligated easily.
- Blunt-end ligation requires a higher DNA concentration than ligation of sticky ends.
DNA transfer into cells
- Transfer of DNA (plasmid or viral vector) into host organisms is done using:
- Transformation: Bacterial cells.
- Transfection: Eukaryotic cells.
Cellular screening
- Used to identify cells containing the DNA of interest.
- A variety of methods are used, including:
- Antibody staining.
- Reporter genes.
Basic molecular techniques
- Molecular biology methods are used to study the molecular basis of biological activity.
- The essence of cell chemistry is to isolate a particular cellular component and then analyze its chemical structure and activity.
- Methods most commonly used to explore cells, their characteristics and processes:
- Nucleic acid methods.
- Protein methods.
- Immunostaining methods.
- Method is the approach or pathway (a process).
- Technique is a man made strategy or tactic (practical aspects).
Molecular biology techniques
- DNA cloning:
- Cut & paste DNA
- DNA or RNA isolation
- Ligation
- Bacterial transformation or transfection
- Chromosome integration
- Expression cloning
- Gel electrophoresis:
- Various types.
- Molecular hybridization:
- Southern blot.
- Northern blot.
- Western blot.
- Reading and writing DNA:
- DNA sequencing.
- DNA synthesis.
- Rewriting DNA: mutations:
- Random mutagenesis.
- Point mutation.
- Chromosome mutation.
- CRISPR/Cas9.
- Polymerase Chain Reaction (PCR):
- DNA polymerase DNA dependent.
- PCR dynamics.
- PCR types.
- Cell culture:
- Cellular screening.
- Arrays:
- DNA array.
- Protein array.
DNA cloning overview
- The inserted DNA is reproduced along with the vector.
- The cloned DNA is copied as the host cell replicates.
- The original DNA is replicated multiple times.
- The recombinant DNA can be inserted into a host cell.
Learning Outcomes
- Define molecular biology methods and techniques.
- Explain DNA cloning techniques.
- Describe DNA and plasmid extraction.
- Describe the CRISPR-Cas9 system.
- Describe DNA synthesis and DNA microarray.
- Explain the different types of DNA mutation.
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Description
This quiz covers essential techniques in molecular biology including chromosome integration, transposon-mediated gene transposition, homologous recombination, and DNA extraction. Each section elaborates on the methods and their significance in genetic manipulation and research.
