Molecular Biology Techniques

Study Notes

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.

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.

A single crossover between a targeting gene and a homologous DNA fragment on a chromosome.
The whole plasmid sequence is integrated.

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.

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.

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.

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.

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.

Transfer of DNA (plasmid or viral vector) into host organisms is done using:
- Transformation: Bacterial cells.
- Transfection: Eukaryotic cells.

Used to identify cells containing the DNA of interest.
A variety of methods are used, including:
- Antibody staining.
- Reporter genes.

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).

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.