Recombinant DNA technology
Producing DNA fragments
Using reverse transcriptase
The enzyme reverse transcriptase is an enzyme that is found in only some viruses and
bacteria and catalyses the formation of a double strand of DNA from a single strand on
RNA. It is found in retroviruses such as HIV. This allows us to make working versions of DNA
that act as genes, by extracting mRNA from cells where that gene is being expressed.
, - a cell that readily produces the protein is selected (for example a beta cell of the
islets of Langerhans from the pancreas are used to produce insulin)
- these cells have large quantities of the relevant mRNA, which is therefore more
easily extracted
- reverse transcriptase is then used to make DNA from RNA. this DNA is known as
complementary DNA (cDNA) because it is made up of the nucleotide that are
complementary to the mRNA
- to make the other strand of DNA, the enzyme DNA polymerase is used to build up
the complementary nucleotides on the cDNA template. this double strand of DNA is
the required gene.
Using restriction endonucleases
Restriction endonucleases are enzymes, extracted from bacteria, that cut DNA at specific
sequences, usually six base pairs in length. The most useful restriction endonucleases are
those that make staggered cuts, as they leave sticky ends on the DNA. The diagram shows
the difference between sticky ends and blunt ends.
Sticky ends are important because if the same restriction endonuclease is used to cut two
DNA fragments, then the ends will be complementary. This allows them to attach together
before stronger covalent bonds form.
Producing DNA fragments
Using reverse transcriptase
The enzyme reverse transcriptase is an enzyme that is found in only some viruses and
bacteria and catalyses the formation of a double strand of DNA from a single strand on
RNA. It is found in retroviruses such as HIV. This allows us to make working versions of DNA
that act as genes, by extracting mRNA from cells where that gene is being expressed.
, - a cell that readily produces the protein is selected (for example a beta cell of the
islets of Langerhans from the pancreas are used to produce insulin)
- these cells have large quantities of the relevant mRNA, which is therefore more
easily extracted
- reverse transcriptase is then used to make DNA from RNA. this DNA is known as
complementary DNA (cDNA) because it is made up of the nucleotide that are
complementary to the mRNA
- to make the other strand of DNA, the enzyme DNA polymerase is used to build up
the complementary nucleotides on the cDNA template. this double strand of DNA is
the required gene.
Using restriction endonucleases
Restriction endonucleases are enzymes, extracted from bacteria, that cut DNA at specific
sequences, usually six base pairs in length. The most useful restriction endonucleases are
those that make staggered cuts, as they leave sticky ends on the DNA. The diagram shows
the difference between sticky ends and blunt ends.
Sticky ends are important because if the same restriction endonuclease is used to cut two
DNA fragments, then the ends will be complementary. This allows them to attach together
before stronger covalent bonds form.
