CIE A Level Biology复习笔记19.1.4 Genetic Engineering: Enzymes

• Genetic engineering is the deliberate modification of a specific characteristic (or characteristics) of an organism. The technique involves removing a gene (or genes), with the desired characteristic, from one organism and transferring the gene (using a vector) into another organism where the desired gene is then expressed
• In order to genetically engineer an organism there are a number of enzymes required:
  • Restriction endonucleases (enzymes) – cuts 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 the specific characteristic because specialised cells make very specific types of mRNA (eg. 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

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