21 Recombinant DNA Technology
Producing DNA Fragments
Techniques have been developed to isolate genes, clone them and transfer them into
microorganisms, which are then grown for the continuous production of a desired protein.
The DNA of two different organisms that have been combined in this way is called recombinant
DNA.
The process of making a protein in this way involves:
1. Isolation - of DNA fragments with gene for desired protein
2. Insertion - of DNA fragment into vector
3. Transformation - transfer of DNA into suitable host cells
4. Identification - of host cells that have successfully taken up DNA
5. Growth/cloning - of host cells to produce protein on larger scale
Isolation
3 ways to produce DNA fragments:
1. Reverse transcriptase
® Locate cell that produces desired protein
® These cells have lots of mRNA, which is extracted
® RT is used to make cDNA from RNA
cDNA is complementary DNA – made up of nucleotides that are complementary to the mRNA
® DNA polymerase is used to build the other DNA strand
® This DNA is the gene needed
2. Restriction endonucleases
® Cut DNA at specific regions – recognition sites
® Creating blunt ends (straight) or sticky ends (staggered)
When you read unpaired bases from left to right, the two sequences are opposites – palindrome
® Viruses inject their DNA into organisms
® In defence, some use RE to cut up the viral DNA
3. Gene machine
® Fragments of DNA can be synthesised from scratch without a pre-existing DNA
template
® Required sequence is designed
® Bioinformatics – nucleotide sequence is fed into computer, which designs a series of
short sections of DNA (oligonucleotides)
® Oligonucleotides are produced and joined together, making larger DNA fragments
Amplifying DNA Fragments
In vivo cloning – copies of the DNA fragment are made inside a living organism
Two restriction fragments can anneal if they have complementary sticky ends, but only by weak
hydrogen bonds.
DNA ligase completes the DNA backbone by forming phosphodiester bonds
Insertion
® The same RE are used to cut the vector (plasmid), producing sticky ends
® The required gene and plasmid will combine as both are complementary
Transformation
® The plasmid is inserted into bacterial cells, along with calcium ions - which make bacteria
permeable
Producing DNA Fragments
Techniques have been developed to isolate genes, clone them and transfer them into
microorganisms, which are then grown for the continuous production of a desired protein.
The DNA of two different organisms that have been combined in this way is called recombinant
DNA.
The process of making a protein in this way involves:
1. Isolation - of DNA fragments with gene for desired protein
2. Insertion - of DNA fragment into vector
3. Transformation - transfer of DNA into suitable host cells
4. Identification - of host cells that have successfully taken up DNA
5. Growth/cloning - of host cells to produce protein on larger scale
Isolation
3 ways to produce DNA fragments:
1. Reverse transcriptase
® Locate cell that produces desired protein
® These cells have lots of mRNA, which is extracted
® RT is used to make cDNA from RNA
cDNA is complementary DNA – made up of nucleotides that are complementary to the mRNA
® DNA polymerase is used to build the other DNA strand
® This DNA is the gene needed
2. Restriction endonucleases
® Cut DNA at specific regions – recognition sites
® Creating blunt ends (straight) or sticky ends (staggered)
When you read unpaired bases from left to right, the two sequences are opposites – palindrome
® Viruses inject their DNA into organisms
® In defence, some use RE to cut up the viral DNA
3. Gene machine
® Fragments of DNA can be synthesised from scratch without a pre-existing DNA
template
® Required sequence is designed
® Bioinformatics – nucleotide sequence is fed into computer, which designs a series of
short sections of DNA (oligonucleotides)
® Oligonucleotides are produced and joined together, making larger DNA fragments
Amplifying DNA Fragments
In vivo cloning – copies of the DNA fragment are made inside a living organism
Two restriction fragments can anneal if they have complementary sticky ends, but only by weak
hydrogen bonds.
DNA ligase completes the DNA backbone by forming phosphodiester bonds
Insertion
® The same RE are used to cut the vector (plasmid), producing sticky ends
® The required gene and plasmid will combine as both are complementary
Transformation
® The plasmid is inserted into bacterial cells, along with calcium ions - which make bacteria
permeable
