Lecture Four - Genetic manipulation Flashcards
How can you clone a gene?
Extract DNA from organism of interest.
Cut the DNA with a restriction enzyme.
Mix the digested DNA with a cloning vector (e.g. plasmid) cut with the same restriction enzyme.
Add DNA ligase.
Transform these recombinant plasmids into bacteria.
Note - transcription enzymes are usually used by bacteria to protect them selves from viruses which insert their DNA into bacteria.
How is the DNA transferred between bacterial cells?
a) Bacterial transformation - important for gene cloning.
Bacteria will only take up DNA if they are ‘compitent,’ this can be induced by treating the bacteria with chemicals.
b) Bacterial transduction.
c) Bacterial conjugation.
What is a genomic library?
A collection of DNA from a particular organism.
The DNA is stored in a population of identical vectors, each containing a different insert of DNA.
In order to construct a genomic library, the organism’s DNA is extracted from cells and then digested with arestriction enzyme to cut the DNA into fragments of a specific size.
The fragments are then inserted into the vector using the enzyme, DNA ligase.
Next, the vector DNA can be taken up by a host organism- commonly a population of Escherichia coli or yeast- with each cell containing only one vector molecule.
Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the libraryfor analysis.
The smaller the molecules of DNA, the faster they migrate.
Thus DNA sequences are separated out based on size.
How is cDNA cloned?
cDNA is a synthetic gene copy without the introns.
How it works:
DNA is extracted from organism.
Reverse transcriptase is used with a cloning vector to create recombinant plasmids (many different ways to do this).
Recombinant plasmids are transformed into bateria.
A cDNA library is created.
A cDNA library contains all the coding regions of all expressed genes from that tissue. Which is helpful, as bacteria cannot splice out introns, therefore this helps to save time.
What is reverse transcriptase?
An enzyme which catalyses the formation of RNA from a DNA template during transcription, or (reverse transcriptase) the formation of DNA from an RNA template in reverse transcription.
Once the DNA is made, transcription and translation can procewed normally using the newly made DNA as the template.
How can reverse transcriptase be used to make cDNA from mRNA?
1) Reverse transcriptase is added to a test tube containing mRNA isolated form a certain type of cell.
2) Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT’s as a DNA primer.
3) mRNA is degraded by another enzyme.
4) DNA polymerase synthesises the second strand, using a primer in the reaction mixture. Several options exist for primers.
5) The result is cDNA, which carries the complete coding sequence of the gene but no introns. YAY!
What is polymerase chain reaction (PCR)?
Used when you want to amplify one section of DNA, or want to make DNA using a known sequence of DNA.
Isolating/amplifying the section of the genome:
1) Isolate the DNA.
2) Design primers that bind to either end of the region of interest.
3) Set up a PCR reaction (needs nucleotides, DNA polyermase etc.).
4) Thermal cycling allows the specific region of DNA to be amplified.
