recombinant DNA Flashcards
how are some disease causes
by individuals unable to make certain protiens
how are protiens created
by the lengths of dna
how were mising protiens fixed previously
the protien was taken from an individual and injected into the host
what issues did this cause
rejection
what has technology allowed
us to isolate the gene , manipulate them and insert into the organism
what is recombinant dna
when the dna from two species are combined forming gmo or transgenic
why can we produce recombinant dna
as the genetic code is universal so transcription and translation can occur in all cells
what are the summary stages
isolation of dna insetion of isolated dna into vector transformation into suitable host identification of sucessful host growth and cloning of succesful host
how can we isolate a gene
by converting mrna to dna using reverse transcriptase
using restriction endonucleases to cut fragmented gene
make gene using the gene machine
what is recognised first
the cell which produces the required protien
what happens to the cell
it is centrifuged to release MRNA
what happens to the mrna
with reverse transcriptase it forms a complimentry dna strand forming a hetro duplex
what happens to this hetroduplex
it is hydrolysed releasing the mrna strand
what happens to the resulting dna strand
the complementry dna numcleotides join using dna polymerase
why do bacteria have restriction endonuclese
to protect them from invading virus they are able to cut the dna at a certain section
what do the enzymes have
active sites that cut as specific recognition sites
how are the recognition sits usiually cut
in a palindrome way with sticky ends at opposite base pairs
what are the two types of ends
blunt or staggered sticky ends
what do these sticky ended fragments contain
the dna for the desired gene
what is determined in the gene machine
the sequences of bases for the desired protien aswell as its amino acid sequence
what triplets are worked out
the complementry dna amino acid triplets
what is done to this sequence
it is fed into a computer
what does the computer design
small sequence of overlapping single nucleotide strands
how are the strands assembled
one base at a time into the correct sequence to make the desired gene
what does the gene contain/ not contain
it only contains extrons
how is the gene replicated
using the polymerase chain reaction
how are the right genes checked
they are scrrened under standard sequencing techniques with errors being rejected
in reverse transcriotase methods the complimentry dna contain only what
exons so is shirter than other dna
what is the strength of in vivo cloning in terms of contamination
less likely to get contaminates as only bases with complimentary base pairing can anneal which the bases are cut using restriction enzymes
what also does it produce
produces an active protein rather than just the gene as the bacteria will transalate the gene in its genome
what is the disadvantage of in vivo
it is time consuming as the gene has to be identified inserted and transformed and then cloes
what else is a weakness in terms of succession
transformation has a very low success rates and recombinace is very low success
what is needed
pure large amount of dna
