Genetic Engineering + Ethics of Genetically Modified
Organisms
Before, only selective breeding allowed to choose desired characteristics for
particular organism (naturally present in population of species interested in *cows*)
Cut out gene of desired characteristic from one organism + transfer it to entirely
different organism (genetically modified organism using recombinant DNA = Re
combined w piece of DNA from 1 organism from other)
EXAMPLE: Insulin into bacteria
1) obtaining required gene (desired DNA fragment)
2) insert gene into vector
3) get vector into recipient cell (plant/animal cell)
4) Identify which cells have been transformed.
5) Allows recipient cell to express gene (getting gene product made by another
organism)
1. obtaining required gene:
3 ways…
1) By polynucleotide synthesiser (making DNA in lab by its constituent parts like DNA
POLYMERASE, FREE NUCLEOTIDES, DNA TEMPLATE
2) use RESTRICTION ENDONUCLEASES (enzymes that cut out gene + specific enzymes that
cut at specific base sequence location; so, particular base sequence = substrate that’s
complementary to active site of restriction endonucleases)
… restriction endonucleases cut in PALINDROMIC (e.g., Hannah) base sequences.
Directionality of palindromic sequence = 5 prime to 3 prime + vice versa
When restriction endonucleases make cuts through palindromic base sequence;
makes sticky ends (overhangs by few bases = complementary to any piece of DNA
that’s been cut using same restriction endonuclease) = useful for genetic engineering
, restriction endonucleases can also cut straight (straight through double helix DNA so
no sticky ends that overhang, instead called BLUNT ends- not much use to us)
sticky ends useful cuz if we have required gene that’s been cut with particular
restriction endonuclease, we cut vector with same restriction endonuclease, then
desired gene (sticky ends) pairs up using complementary base pairing forming
hydrogen bonds between overhanging ends = recombinant DNA
3) mRNA -------------------------------------- cDNA (small c = complementary)
Restriction transcriptase
required gene can be obtained by isolating mRNA from cells that express gene of
interest.
Use enzyme that reverse transcribes mRNA back into DNA (transcription = DNA into
mRNA).
Mrna = single stranded, cDNA = single stranded
ADVANTAGES of using Mrna method:
1. Loads of Mrna.
2. Mrna does contain introns.
DISADVANTAGES of using Mrna method:
1. Undergo another step adding DNA polymerase so single stranded
complementary DNA can be used as template strand (like in DNA
REPLICATION) to produce double stranded DNA because double stranded
DNA is the gene.
2. Inserting gene into vector
Vector = carries gene between organisms (bacteria, plasmid, virus)
1) Cut BOTH required gene + vector with SAME restriction endonuclease
2) Combine with DNA ligase (enzyme that can join up single strands of double stranded DNA
to another piece of double stranded DNA)
3) Makes recombinant DNA
3. Getting vector into recipient cell
Organisms
Before, only selective breeding allowed to choose desired characteristics for
particular organism (naturally present in population of species interested in *cows*)
Cut out gene of desired characteristic from one organism + transfer it to entirely
different organism (genetically modified organism using recombinant DNA = Re
combined w piece of DNA from 1 organism from other)
EXAMPLE: Insulin into bacteria
1) obtaining required gene (desired DNA fragment)
2) insert gene into vector
3) get vector into recipient cell (plant/animal cell)
4) Identify which cells have been transformed.
5) Allows recipient cell to express gene (getting gene product made by another
organism)
1. obtaining required gene:
3 ways…
1) By polynucleotide synthesiser (making DNA in lab by its constituent parts like DNA
POLYMERASE, FREE NUCLEOTIDES, DNA TEMPLATE
2) use RESTRICTION ENDONUCLEASES (enzymes that cut out gene + specific enzymes that
cut at specific base sequence location; so, particular base sequence = substrate that’s
complementary to active site of restriction endonucleases)
… restriction endonucleases cut in PALINDROMIC (e.g., Hannah) base sequences.
Directionality of palindromic sequence = 5 prime to 3 prime + vice versa
When restriction endonucleases make cuts through palindromic base sequence;
makes sticky ends (overhangs by few bases = complementary to any piece of DNA
that’s been cut using same restriction endonuclease) = useful for genetic engineering
, restriction endonucleases can also cut straight (straight through double helix DNA so
no sticky ends that overhang, instead called BLUNT ends- not much use to us)
sticky ends useful cuz if we have required gene that’s been cut with particular
restriction endonuclease, we cut vector with same restriction endonuclease, then
desired gene (sticky ends) pairs up using complementary base pairing forming
hydrogen bonds between overhanging ends = recombinant DNA
3) mRNA -------------------------------------- cDNA (small c = complementary)
Restriction transcriptase
required gene can be obtained by isolating mRNA from cells that express gene of
interest.
Use enzyme that reverse transcribes mRNA back into DNA (transcription = DNA into
mRNA).
Mrna = single stranded, cDNA = single stranded
ADVANTAGES of using Mrna method:
1. Loads of Mrna.
2. Mrna does contain introns.
DISADVANTAGES of using Mrna method:
1. Undergo another step adding DNA polymerase so single stranded
complementary DNA can be used as template strand (like in DNA
REPLICATION) to produce double stranded DNA because double stranded
DNA is the gene.
2. Inserting gene into vector
Vector = carries gene between organisms (bacteria, plasmid, virus)
1) Cut BOTH required gene + vector with SAME restriction endonuclease
2) Combine with DNA ligase (enzyme that can join up single strands of double stranded DNA
to another piece of double stranded DNA)
3) Makes recombinant DNA
3. Getting vector into recipient cell
