Gene Isolation Methods Flashcards
Why is gene isolation important in molecular biology
Gene isolation allows scientists to study, modify, or transfer specific genes by identifying and replicating their DNA at high copy numbers in host cells or in vitro.
What is a major challenge when isolating genes for replication in a host?
The DNA must contain a replication origin to be replicated in a host.
What is the second main challenge of gene isolation?
Genomes have many genes—identifying the specific gene of interest is difficult.
How can vectors help solve gene isolation challenges?
Vectors (like plasmids) are replicons, meaning they contain a replication origin and can be used to clone and replicate a single DNA fragment in a host.
How does creating a DNA library help gene identification?
You can create a library of host cells, each containing a different DNA fragment. The gene of interest can be identified through screening.
What is an alternative to in vivo replication in gene isolation?
Polymerase Chain Reaction (PCR) replicates DNA in vitro at high copy numbers, allowing for faster, non-cell-based DNA amplification.
What are the essential components required for gene cloning?
1) A vector (often a plasmid), 2) DNA fragments, 3) A host organism (commonly E. coli).
What is recombinant DNA?
A molecule formed by combining a plasmid vector and a foreign DNA fragment.
What happens to recombinant plasmids once introduced into a host?
They replicate, and host cells form colonies, each carrying many copies of the recombinant DNA.
What does a library of colonies represent?
A population of cells where each colony contains a different DNA fragment.
What must be done if all colonies are different?
The library must be screened to find the one containing the gene of interest.
What are restriction endonucleases and who discovered them?
Enzymes that cut DNA at specific sequences. Discovered by Linn and Arber (1968), who found they helped bacteria resist phage infection.
How do bacteria protect themselves from their own restriction enzymes?
They methylate their own DNA using a methylase to prevent self-digestion.
What problem did early endonucleases present for cloning?
They cut DNA away from their recognition site, making them less useful.
What are Type II restriction enzymes and why are they important?
They cut at specific palindromic recognition sites, making them predictable and ideal for cloning.
How does recognition site length affect cutting frequency?
- 4 bp cutters cut more frequently (more sites).
6–8 bp cutters cut less frequently.
What are the two types of DNA ends created by restriction enzymes?
Cohesive (sticky) ends: Have overhangs with exposed bases that can hydrogen bond.
Blunt ends: No overhangs, harder to ligate.
Describe the two-step process of DNA fragment ligation.
Hydrogen bonding between cohesive ends.
Formation of phosphodiester bonds by DNA ligase in an ATP-dependent reaction.
How do we monitor DNA manipulation and fragment size?
With agarose gel electrophoresis: DNA is loaded into a gel, and under an electric field, fragments migrate toward the anode (positive).
Why do smaller DNA fragments migrate further in gel electrophoresis?
They more easily penetrate the gel matrix, allowing them to move faster and farther.
What is ethidium bromide used for?
It intercalates between DNA bases and fluoresces under UV, allowing visualization of DNA.
What is a DNA ladder and why is it used?
A commercially purchased set of fragments of known sizes, used as a size reference for unknown DNA fragments.
When would you use a genomic library?
When studying entire transcription units (promoter, exons, introns, terminators) or non-coding/intergenic regions.
How is genomic DNA prepared for a library?
DNA is partially digested with restriction enzymes or randomly sheared to form large overlapping fragments.
