recombinant DNA technology Flashcards
what is recombinant dna technology
involves transfer of fragments of DNA from one organsim/ species to another
wht is the DNA of two or more organisms that has been combined called
recombinant DNA
why is it possible
genetic code, transcription and translation machinary universal between all species,
trnsfered DNA fragemtns can be translated within cells of recipient organism (transgenic/GMO)
stages of making a protein
isolation
insertion
transformation
identification
growth/cloning
3 ways to form/ isolate DNA fragments
reverse transcriptase
restriction endonucleases
gene machine
reverse transcriptase
protein producing cell with large amounts of mRNA selected
reverse transcriptase uses mRNA as template to produce cDNA
cDNA isolated by hydrolysis of mRNA by enzyme
double stranded DNA formed on the template cDNA using DNA polymerase
copy of desired gene formed
does cDNA have introns
no, mRNA doesnt have introns
where do restriction endonucleases cut
recognition sequence (specific base sequence)
gene machine order
protein > amino acid order > mRNA codons > DNA
gene machine
DNA sequence etered into computer
checked for biosecurtiy, biosafety and ethics
computer designs oligonucleotides
computer build up oligonucleotides by joining individual nucleotides together
oligonucleotides joined to make a gene
DNA fragments amplified ( PCR or in vitro)
to make double strands and then replicate gene
gne insterted into bacterial plasmid which can replicate and transfer gene
new gene checled for mistakes
bacteria cells contianing gene cloned
PCR
double stranded DNA fragments heated to 95, H bonds between base pairs break producing two single DNA strands
cooled to 55, allow primers to anneal to DNA fragments
heated to72, opt temp for DNA polymerase
DNA polymerase attaches nucleotides together to from 2 new double stranded DNA fragments
what goes into PCR
DNA fragments
DNA polymerase
primers
nucleotides
thermocyclers
number of dna fragments produced by a given nuber of cycles=
2^x
x= number of cycles
(2 fragments produced pe crycle)
in vivo- transformation
DNA cut using restriction endonuclraases to create sticky ends
promoter and terminatoer region added to allow transcripton
same restirction endonuclease is used to cut open the plasmid to allow complimentary sticky end
enzyme: DNA ligase used to incorporate DNA fragment into plasmid
recombinant DNA formed
recombinant plasmid transferred into bacteria via heat shock and Ca2+
how can we identify which bacterial cells have taken up recombinant plasmids
marker genes
why are marker genes useful
not all plasmids take up foreign DNA fragments
not all recombinant plasmids will be taken up by bacteria cells
identify if plasmid was taken up
all bacterial cells grown on medium that contains antiobiotic ampicillin
bacterial cells that have taken up plasmid have acquired the gene for ampicillan resistance
these bacterial cells break down ampiciilain and survive
bacterial cells that have not taken up plasmids will not be reistant-die
identify if recombiannt palsmids are taken up
insert DNA fragment into maker gene
if function is performed, recombinant DNA has not been taken up
if function is not performed, recombinant DNA has been taken up
identify if plasmid was taken up
all bacterial cells grown on medium that contains antiobiotic ampicillin
bacterial cells that have taken up plasmid have acquired the gene for ampicillan resistance
these bacterial cells break down ampiciilain and survive
bacterial cells that have not taken up plasmids will not be reistant
how to identify living colonies of bacteria containing required gene
replica plating
gene therapy
mechanism by which genetic disorders can be cured or treated by masking thr effect of the fualty allele with the insertion of the functional allele
