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What does recombinant DNA technology involve?
the transfer of fragments of DNA from one organism, or species, to another
What is recombinant DNA?
the DNA of two different organisms that have been combines
The resulting organism from recombinant DNA can be referred to as what?
transgenic
or
GMO - genetically modified organism
Since the genetic code is universal what does this mean for the transgenic organism?
the transcription and translation mechanisms are the same
the transferred DNA can be translated within cells of the recipient
What are the 3 ways fragments of DNA can be produced?
mRNA to cDNA using reverse transcriptase
using restriction enzymes to cut a fragmnet
creating the gene in a gene machine
What are the 5 key stages of producing transgenic organisms?
isolation of DNA fragments
insertion of DNA fragments into a vector
transformation - transfer of DNA into suitable host
Identification of host cells that have successfully taken yp the gene
growth/cloning
How is mRNA converted to cRNA?
mRNA isolated (cells are centrifuged)
free DNA nucleotides are added
reverse transcriptase joins DNA nucleotides to mRNA which acts as a template
enzyme (ribonuclease H) hydrolyses mRNA to get cRNA
is cRNA double or single-stranded?
single-stranded
What happens once we have cDNA?
DNA polymerase catalyses the formation of a complementary strand of DNA
produced a double strand (using free DNA nucleotides from earlier)
a copy of the gene now exists, with no introns
How are the cells chosen?
cell that readily produces the protein is selected
cells have large quantities of relevant mRNA which is more easily extracted
What do restriction endonuclease do?
cut up a DNA strand at a specific sequence of bases called a recognition sequence
Recognition sites are usually what?
palindromic (read the same left to right and right to left)
What are the different ways restriction endonuclease can cut up DNA?
some make clean cuts straight down - blunt ends
some make staggered cuts - sticky ends
What are oligonucleotides?
single strands of DNA
how does a gene machine work?
desired sequence of nucleotides can be determine by primary structure of protein
desired nucleotide base sequence is fed into a computer
the computer designs series of overlapping oligonucleotides which can be assembled into the desired gene
automated process assembles each of the oligonucleotides
oligonucleotides are joined together to make a gene - amplified by PCR
What happens once the gene is made?
using sticky ends, the gene can be inserted into a bacterial plasmid to be eventually cloned
Why can’t restriction enzymes be used to produce DNA that E.coli could be used to produce a human gene?
human DNA contains introns
E.coli cannot remove introns
Why is it better to use the gene machine than reverse transcriptase to convert mRNA into cDNA?
faster to use gene machine than enzyme-controlled reactions