Molecular Techniques Flashcards
What are some of the uses of recombinant DNA technology?
Expression and purification of target proteins, tagging and expression of proteins for visualization, improving traits of crops and animals.
How is recombinant DNA technology possible?
The genetic code is universal - codons code for individual amino acids. A gene from one organism can be expressed in another.
How do we clone?
- Extract gene from template
2. Use PCR (denaturation, annealing and synthesis)
Where are the genes for cloning going to come from?
If source is bacterial use PCR. If the source is human need to remove the introns before translation. BUT we cant PCR from mRNA so must reverse transcribe mRNA back to DNA.
How do we make cDNA?
Physical card 35.
What are the basic requirements for getting a cell to make your product?
A promoter, target protein coding sequence.
What is the requirement for a promoter for expressing a target sequence?
Must be the appropriate promoter e.g. mouse retinal promoter and green fluorescent protein coding sequence to make green eyes.
How do you get a cell to produce your gene of interest and pass it onto future generations?
Have to use plasmids in bacterial cells - capable of self-replication, selection pressure and are transferable.
What is the process of having a cell take in and express the protein?
Obtain a plasmid from bacterial cells. Make a cut to fuse the two pieces of DNA together and propagate the bacterial cell. Physical card 36.
What is a recombinant molecule?
Target DNA and Vector DNA joined via restriction enzymes making space in the DNA - gaps in phosphate backbone are repaired by ligase.
What is a transformant cell?
A cell that is carrying the plasmid.
How do we distinguish transformants from non transformants.
Use antibiotic resistant genes in plasmids. Put cells onto a plate where bacteria can grow - only cells carrying plasmid can grow in the plate that can grow on the plate containing antibiotics.
What are recombinant molecules?
Are carrying the gene of interest after distinguishing transformants.
How do we distinguish recombinant molecules?
Put a multiple cloning site in another gene ( a reporter gene as a screen able marker of the multiple cloning site). E.g. LacZ gene. In non-recombinant Lac Z functional and X gal (colourless compound added) is cleaved and becomes blue. In recombinant no cleave and stays white.
What are some screening methods other than Blue/White selection?
Colony PCR
Restriction digestion
Sequencing
