Genetic Engineering: Enzymes
- In order to genetically engineer an organism there are a number of enzymes required:
- Restriction endonucleases (enzymes) – cut the DNA strands so that the desired gene can be isolated or spliced (inserted) into a vector
- Reverse transcriptase – reverses transcription to produce a single-strand complementary DNA (cDNA) from an mRNA strand with the code for the desired gene
- DNA polymerase – used to convert the single-stranded cDNA into a double-stranded DNA molecule of the desired gene
- DNA ligase – is used to splice (insert) the gene into the vector
Restriction endonucleases
- The role of restriction endonucleases (or restriction enzymes) in the transfer of a gene into an organism is to:
- Isolate the desired gene
- Separate the DNA strands (at the same base sequence) in a vector so the desired gene can be inserted
- There are many different restriction endonucleases because they bind to a specific restriction site (specific sequences of bases) on DNA, eg. HindIII will always bind to the base sequence AAGCTT
- Restriction endonucleases will separate the two strands of DNA at the specific base sequence by ‘cutting’ the sugar-phosphate backbone in an uneven way to give sticky ends or straight across to give blunt ends
- Sticky ends result in one strand of the DNA fragment being longer than the other strand
- The sticky ends make it easier to insert the desired gene into another organism's DNA or into a vector as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA that have been cut with the same restriction endonucleases
Reverse transcriptase
- The role of reverse transcriptase in the transfer of a gene into an organism is to produce a single-strand complementary DNA molecule (cDNA) that contains the code for the desired characteristic, this will then be inserted into a vector (after being converted into a double-stranded DNA molecule)
- Reverse transcriptase enzymes are sourced from retroviruses and they catalyse the reaction that reverses transcription. The mRNA (with the genetic code for the desired gene) is used as a template to synthesise a single strand of complementary DNA (cDNA)
- Reverse transcriptase enzymes are often used as it is easier for scientists to find mRNA with specific characteristics because specialised cells make very specific types of mRNA (e.g. β-cells of the pancreas produce many insulin mRNA) and mRNA does not contain introns
DNA polymerase
- DNA polymerase is used to convert the single strand of cDNA into a double-stranded DNA molecule which contains the desired code for the gene
- The enzyme builds the second strand by pairing free nucleotides with the complementary bases on the cDNA strand, exactly like in normal DNA replication
DNA ligase
- DNA ligase catalyses the formation of phosphodiester bonds in the DNA sugar-phosphate backbone
- This enzyme enables the isolated desired gene to be spliced into a vector (generally a plasmid) so that it can be transferred to the new organism
Examiner Tip
It is essential you use the names of these enzymes when you are explaining genetic engineering. You should also refer to ‘sticky ends’ when discussing the role of restriction endonucleases. Remember the same restriction endonuclease must be used in isolating the desired gene and in separating the DNA in the vector. It is also important to state that the restriction enzymes cut the DNA or plasmid NOT the gene.
