Producing DNA Fragments Flashcards
In Vivo Cloning: Using Restriction Endonucleases
> Cut DNA in the middle of a recognition sequence that is palindromic
> Produces blunt / sticky ends, in cloning the most important thing is an uneven cut, so each strand of DNA is exposed
> The same restriction endonuclease is used to cut DNA so all fragments have complementary ends, the single stranded end can be stuck onto the single stranded end of any fragment
> This allows the DNA to be joined together in a vector using DNA ligase (forms the sugar phosphate backbone) to join the nucleotides together
In Vivo Cloning: Conversion of mRNA into cDNA using reverse transcriptase
> Extract mRNA from cytoplasm of B-cells in the pancreas
> Create a single strand of cDNA from mRNA using reverse transcriptase
> Create double stranded cDNA using DNA nucleotides and DNA polymerase
> Use a PCR machine to make many copies of cDNA for insulin
> Advantage: cDNA is intron free because it is based on the mRNA template
In Vivo Cloning: The Gene Machine
> Identify protein you want to clone & examine
> Identify amino acid sequence - work out mRNA and DNA sequences
> DNA sequence is put into computer which checks biosafety & biosecurity ( is the DNA being created safe & ethical? )
> Computer creates small sections of overlapping single strands of nucleotides - then joined to create the DNA for the entire gene
> PCR used to amplify quantity
> Advantages = quick, accurate, intron free
