International A Level Biology / Gene technology
Producing recombinant DNA revision notes
Edexcel International A Level Biology (YBI11), chapter 25: Gene technology, specification point 8.18. Free lesson.
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Recombinant DNA is DNA joined from two sources, such as a human gene inserted into a bacterial plasmid. Restriction enzymes, complementary sticky ends and DNA ligase allow the gene to be joined into a plasmid.
How restriction endonucleases cut DNA
A restriction endonuclease is a bacterial enzyme that cuts inside a DNA molecule. Bacteria use these enzymes against viral DNA. Each enzyme recognises a particular base sequence, called its recognition site, which is usually 4 to 8 base pairs long. The site fits the enzyme's active site, so changing a base can stop the enzyme cutting there. The enzyme hydrolyses one phosphodiester bond in the sugar-phosphate backbone of each DNA strand. Many enzymes cut the two strands at different positions. This staggered cut leaves short runs of unpaired bases called sticky ends. Sticky…
How EcoRI makes sticky ends
EcoRI, an enzyme from E. coli, recognises the six-base-pair sequence GAATTC. This sequence is palindromic: read in the 5′ to 3′ direction, each strand has the same sequence. EcoRI cuts between G and A on each strand, at positions that leave four unpaired bases on each piece. The sticky ends each have the sequence AATT. An AATT end can pair with another AATT end, so ends cut by EcoRI are complementary even if they came from different DNA molecules.
Two ways to obtain the gene
You can cut a gene out of DNA or make a DNA copy from mRNA. Cutting uses a restriction endonuclease. The mRNA method makes cDNA, meaning complementary DNA, using reverse transcriptase and then DNA polymerase. For the mRNA method, use cells that make the protein, as they contain lots of its mRNA. For example, pancreatic beta cells make insulin. A gene cut from DNA still contains introns, which are sections that do not code for a protein. The mRNA has already been spliced, so cDNA made from it has no introns. This matters because bacteria cannot remove introns. A gene can be one among…
Joining the gene into a plasmid
A plasmid is a small circle of bacterial DNA. It acts as a vector, carrying the gene into a cell. The plasmid and DNA containing the gene are cut with the same restriction enzyme so they have matching, complementary sticky ends. The enzyme cuts the plasmid at two sites, removing a section, and cuts the gene out of its DNA. The sticky ends pair by hydrogen bonds between complementary bases. This leaves a gap in each sugar-phosphate backbone. DNA ligase closes the gaps by forming phosphodiester bonds in the backbones. The result is one continuous circle called a recombinant plasmid.
Keep the two types of bond distinct
Complementary bases on sticky ends pair by themselves, forming hydrogen bonds between the strands. Ligase does not make these bonds. It joins nucleotides along each strand by forming phosphodiester bonds, sealing the sugar-phosphate backbones. Using the same restriction enzyme gives the plasmid and gene matching sticky ends, so they can pair and then be joined by ligase.