32. Gene editing and DNA manipulating Flashcards
What does denaturing/melting DNA refer to?
Separating double stranded DNA into single strands
What does renaturing/hybridisation refer to?
Means joining single stranded nucleic acids into double strands.
What are the two molecular forces that need to be broken for the denaturing of DNA?
- Hydrogen bonds between complementary bases
2. Stacking of hydrophobic Nitrogenous bases down the centre of the helix
What are the two means by which DNA can be denatured?
- High temperature, >80
2. Chemical, strong alkali
What are the two means by which DNA can be renatured?
- Low temperature, less than 80
2. Chemical, neutral pH
What is the difference between renaturing and hybridisation?
Renaturing refers to DNA - DNA strands combining
Hybridisation refers to either RNA of DNA from different sources combining with another single stranded DNA
What components are needed for PCR?
- Template or original target DNA
- Oligonucleotide primers
- Deoxyribonucleotide triphosphates (dNTP) building blocks
- DNA polymerase
What are the 3 steps of the PCR reaction?
- Denaturing
- Annealing/hybridisation of primers
- Extension/elongation by DNA polymerase
What are the temperatures for each stage of the PCR cycle?
95, 45-65, 70
What are restriction enzymes and recognition sequences?
- Bacteria produce restriction enzymes which can cut DNA at specific sequences
- All restriction enzymes recognise a specific base sequence in the DNA called the recognition sequence
What type of restriction enzymes are used in the lab?
Type II which have a predictable recognition sequence
What do the type 5 restriction enzymes do?
Cas9-gRNA complex for example, uses RNA to target specific sequences.
What mechanisms are in place to ensure restriction enzymes do not self recognise bacterial DNA?
- Restriction enzymes needed to cleave incoming phage DNA
- Methyl groups at restriction sites on the bacterial DNA block the restriction enzyme and protect the bacterial DNA from being cleaved
What DNA can the ends be joined to depending on if they are sticky or blunt?
- Blunt ends can be joined with any blunt end
- Sticky ends can only be joined if the base pairs sticking out match (complementary)
What are isoschizomers?
Different restriction enzymes that recognise the same sequence AND cut similarly
What are neoschizomers?
Different restriction enzymes that recognise the same sequence BUT cut differently
What charge is there on DNA?
DNA is negatively charged due to the negative phosphate group and will migrate from negative to the positive electrode
What are the 4 factors which the gel electrophoresis migration rate depends on?
- Gel concentration: movement is slower in denser gel
- Voltage across field: Slower with lower voltage
- Buffers used
- Conformation: faster rate if DNA is more compact
How can genomic variations be detected with restriction enzymes? (RFLP)
- Variations in genome may lead to loss/gain of restriction enzyme sit: restriction fragment length polymorphism (RFLP)
- If an RFLP is linked (close to) a gene mutation the RFLP will tend to be co-inherited with one of the gene alleles
What are the four components needed for gene cloning?
- DNA
- Vector to carry the different length DNA (plasmid)
- Restriction enzymes and DNA ligase (cut/join recombinant DNA)
- Host cell in which vectors replicate (bacteria)
Why are bacterial plasmids chosen as the most commonly used vector for gene cloning?
- Origin of replication
- Antibiotic resistance for selection
- Multiple cloning sites containing unique restriction enzyme sites
How is the host cell treated to increase uptake of recombinant DNA?
Transformation involves heat shock at 42 degrees to make the membrane porous to uptake DNA and electroporation
What are two ways in which host cells can be screened to see which ones have taken up the plasmid?
- Antibiotic resistance screening: only hosts containing the vector can grow
- LacZ screening only hosts containing the vector can make blue colonies
