Recombinant DNA technology
Recombinant DNA is when DNA of two organisms is combined, creating a transgenic/
genetically modified organism. This is possible due to the universal nature of DNA and
transcription/translation processes.
In order to create recombinant DNA, there are five stages:
1. Isolation of a DNA fragment
2. Insertion of a DNA fragment into a vector
3. Transformation (transfer of DNA into a host cell)
4. Identification of the transgenic host cells
5. Cloning of the transgenic host cells
Production of DNA fragments
Reverse transcriptase
Reverse transcriptase can convert mRNA into DNA. This is useful when producing and isolating
DNA fragments because there are many copies of mRNA in the cytoplasm due to transcription
so they are accessible, but the very few copies of DNA are contained in the nucleus.
1. ISOLATION OF TARGET mRNA
A cell that produces the desired protein is selected and the relevant sections of mRNA are
extracted.
2. USE OF REVERSE TRANSCRIPTASE
The mRNA acts as a template for free DNA nucleotides to form a complementary strand of
cDNA (copy DNA). Reverse transcriptase catalyses the formation of the cDNA strand.
3. FORMATION OF DOUBLE STRANDED cDNA
The cDNA is isolated by the use of an enzyme to hydrolyse the mRNA strand. This produces
single-stranded cDNA. Free DNA nucleotides then bind to the cDNA to form a double stranded
section of cDNA. The formation of phosphodiester bonds between adjacent nucleotides is
catalysed by DNA polymerase.
This creates a double stranded
section of cDNA identical to the
DNA strand that the mRNA was
created from.
, Restriction endonucleases
Restriction endonucleases each cut a DNA strand at a specific recognition sequence. These
tend to be palindromic e.g. AGGCCT.
● STICKY ENDS
The cut made by the restriction endonuclease
is in a different place on each DNA strand,
creating an overlapping area on
single-stranded DNA (see diagram). This is
preferred because it creates a stronger bond
when the fragment is inserted into the vector.
● BLUNT ENDS
The cut made by the restriction endonucleases
is in the same location on both strand, so there
is no overlap. This is less common because it
causes a weaker bond when the fragment is
inserted into the vector.
Recombinant DNA is when DNA of two organisms is combined, creating a transgenic/
genetically modified organism. This is possible due to the universal nature of DNA and
transcription/translation processes.
In order to create recombinant DNA, there are five stages:
1. Isolation of a DNA fragment
2. Insertion of a DNA fragment into a vector
3. Transformation (transfer of DNA into a host cell)
4. Identification of the transgenic host cells
5. Cloning of the transgenic host cells
Production of DNA fragments
Reverse transcriptase
Reverse transcriptase can convert mRNA into DNA. This is useful when producing and isolating
DNA fragments because there are many copies of mRNA in the cytoplasm due to transcription
so they are accessible, but the very few copies of DNA are contained in the nucleus.
1. ISOLATION OF TARGET mRNA
A cell that produces the desired protein is selected and the relevant sections of mRNA are
extracted.
2. USE OF REVERSE TRANSCRIPTASE
The mRNA acts as a template for free DNA nucleotides to form a complementary strand of
cDNA (copy DNA). Reverse transcriptase catalyses the formation of the cDNA strand.
3. FORMATION OF DOUBLE STRANDED cDNA
The cDNA is isolated by the use of an enzyme to hydrolyse the mRNA strand. This produces
single-stranded cDNA. Free DNA nucleotides then bind to the cDNA to form a double stranded
section of cDNA. The formation of phosphodiester bonds between adjacent nucleotides is
catalysed by DNA polymerase.
This creates a double stranded
section of cDNA identical to the
DNA strand that the mRNA was
created from.
, Restriction endonucleases
Restriction endonucleases each cut a DNA strand at a specific recognition sequence. These
tend to be palindromic e.g. AGGCCT.
● STICKY ENDS
The cut made by the restriction endonuclease
is in a different place on each DNA strand,
creating an overlapping area on
single-stranded DNA (see diagram). This is
preferred because it creates a stronger bond
when the fragment is inserted into the vector.
● BLUNT ENDS
The cut made by the restriction endonucleases
is in the same location on both strand, so there
is no overlap. This is less common because it
causes a weaker bond when the fragment is
inserted into the vector.
