The use of restriction endonucleases and ligases to insert fragments of…

The idea

The same restriction endonuclease cuts both the target DNA and the plasmid vector. The enzyme's active site is complementary to a specific palindromic recognition sequence, hydrolysing phosphodiester bonds in the sugar-phosphate backbone. A staggered cut leaves sticky ends: short single-stranded runs of unpaired bases. Because the same enzyme is used, sticky ends on the fragment are complementary to those on the opened plasmid. When mixed, exposed bases pair and hydrogen bonds form between them. This holds the fragment in place but does not join the strands. DNA ligase then catalyses the formation of phosphodiester bonds between adjacent nucleotides, sealing the backbones to produce a recombinant plasmid.

What earns the marks

• Using a restriction endonuclease to cut the DNA at a specific palindromic recognition sequence.
• Using the same restriction enzyme on the vector, so that complementary sticky ends are produced.
• Complementary bases on the sticky ends pairing, with hydrogen bonds forming between them.
• DNA ligase joining the sugar-phosphate backbones by catalysing the formation of phosphodiester bonds.
• Describing the product as recombinant DNA or a recombinant plasmid.

Common mistakes and how to put them right

• Using different restriction enzymes on the gene and the plasmid and still expecting the ends to anneal.

  Put it right: Specify the same enzyme must be used.

• Saying DNA ligase forms hydrogen bonds.

  Put it right: Ligase catalyses phosphodiester bonds, while hydrogen bonds form spontaneously between complementary bases.

• Saying the restriction enzyme cuts in the middle of the gene.

  Put it right: State that enzymes cut at chosen recognition sites which must flank the target gene to avoid destroying it.

• Stopping at base pairing and never mentioning ligase.

  Put it right: Include ligase to seal the sugar-phosphate backbone.

Exam tips

• Two enzymes, two different bonds: restriction endonuclease breaks phosphodiester bonds, ligase forms them. Say which each time.
• The word 'same' before 'restriction enzyme' is crucial; without it the complementary sticky ends have no explanation.
• Remember that using a single restriction enzyme to cut both ends allows the fragment to insert in either orientation.