Lecture 33- Gene Editing Flashcards
Differ between renaturing and hybridisation.
Renaturing: refers to DNA-DNA strands combining
Hybridisation: refers to either RNA or DNA combining with another single stranded DNA
Describe the mechanism of denaturing and how it differs from renaturing.
Denaturing refers to the separation of double stranded DNA into single strands. This require the breaking of the hydrogen bond between complementary bases as well as the intramolecular attracting caused by the stacking of the bases. This breaking is achieved by either heating or involve the use of strong alkali.
Renaturing differs as it refers to the combination of DNA strands. It is done by using low temperature or neutral chemicals.
The steps of Polymerase Chain Reaction is temperature controlled, including the annealing step. What is the main factor in determining the temperature used for this step?
It depends on the ratio between C and G bases in the target DNA sequence. The more C-G pairing, the higher energy is required to break the bonds (as C-G pairing have three hydrogen bonds, compared to two for A-T)
What is the function of restriction enzymes?
Restriction enzymes can cut DNA at specific sequences by recognising the recognition sequence
Type II restriction enzymes can cut DNA in three different ways. What are they?
3’ overhang, 5’ overhang (sticky ends), blunt ends
What are isoschizomers and neoschizomers?
Both describes enzymes that recognise the same recognition sequence. If they cut the sequence similarly, they’re called isoschizomers. If not, they’re called neoschizomers.
Mention 4 factors which affects the migration rate of DNA fragments in gel electrophoresis.
- Gel concentration
- Voltage across field
- Buffers used
- Conformation
Explain how restriction enzyme digest and gel electrophoresis can detect genome variations.
Genome variations may lead to loss or gain of restriction enzyme site (recognition sequence). This addition/deletion would lead to gene fragments of differing length from “normal” gene sample. This concept is called restriction fragment length polymorphism (RFLP). If RFLP is detected, it can pinpoint to the exact cause of genome variations.
Mention the components required for gene cloning. (4)
DNA, vector, restriction enzymes (including DNA ligase), host cell
How do one screen for hosts that have successfully transformed (take up DNA)?
- Antibiotic resistance screening: only hosts containing the vector an grow e.g. ampicillin resistance
- LacZ screening: only hosts containing the vector can make white colonies. unsuccessful transformations will lead to blue colonies.
What is a vector? What are some of its components?
Vector carries the sample DNA to be cloned in the host cell. Three key features of vectors are the origo, antibiotic resistance sequence, and multiple cloning sites.
What are the key steps involved in amplifying plasmids?
The restriction enzyme will cut the plasmid in the cloning site. This will open up the plasmid, at which the sample DNA (with complementary sticky ends) will be inserted tot he plasmid with the help of the enzyme ligase. However, to assist the uptake of foreign DNA tot he plasmid (transformation process), there are two things than can be done, either heat shock or electroporation.
Why do some competent host are not successfully transformed?
When the restriction enzyme cuts the plasmid in the cloning site, there is a probability that DNA ligase would just bind the ends of the original plasmid without adding the foreign DNA sequence.
