A population of E. coll cells that, together, contain all of the fragments of the genome of Bacterium X
Removing Introns from Eukaryotic Genes Most eukaryotic genes have intervening sequences, or introns, that are removed from precursor messenger RNA by the eukaryotic cell machinery before the mRNA is translated into protein. This presents a problem when genes of eukaryotic cells are cloned in bacteria, because bacteria have no means to remove introns. To overcome this obstacle, the eukaryotic genes are not inserted directly into the vector. Instead, mRNA from which the eukaryotic cell has already removed the introns is first isolated from the appropriate eukaryotic tissue. Then, a strand of DNA complementary to the mRNA is synthesized in vitro using reverse transcriptase, an enzyme encoded by retroviruses. That single strand of DNA is then used as a template for synthesis of its complement, creating double-stranded DNA. The resulting copy of DNA, or cDNA encodes the same protein as the original DNA, but it lacks the introns (figure 9.13). ■ introns, p. 180 ■ retrovirus, p. 356
Using Restriction Enzymes to Generate Fragments of DNA
The purified DNA is cut into smaller fragments by treating it with a restriction enzyme. Hundreds of different restriction enzymes have been discovered. Each enzyme has been given a seemingly peculiar name, but the name simply represents the bacterium from which the enzyme was initially isolated. The first letter represents the first letter of the genus name, the next two letters are derived from the species name; any other numbers or letters designate the strain and order of discovery. For example, a restriction enzyme from E. coli strain RY13 is called EcoRI and an enzyme from Staphylococcus aureus strain 3A is called Sau3A.
Most restriction enzymes recognize a sequence that is a palindrome. In other words, the sequence of one strand is identical to the sequence of the complementary strand when each is read in the 5' to 3' direction. Recall that the two DNA strands are antiparallel; thus, they are read in opposite directions. The restriction enzyme recognizes and cuts the identical sequence on each strand of the double-stranded molecule. The sequence that a particular enzyme recognizes is called a recognition sequence (table 9.3). ■ antiparallel, p. 168
Cutting, or digesting, DNA with a restriction enzyme generates either a sticky or a blunt end, depending on where the enzyme cuts in the sequence. An enzyme such as AluI that cuts in the middle of the recognition sequence generates blunt ends, whereas an enzyme such as BamHI that produces a staggered cut generates ends with a short overhang of usually four bases. The overhangs are called sticky ends or cohesive ends because they will form base pairs, or anneal, with one another. Because any two complementary cohesive ends can anneal, regardless of the source of DNA, these ends can be used to facilitate the joining of DNA fragments from different sources (figure 9.14).
Figure 9.12 A DNA Library Each cell contains one fragment of a given organism's genome.
Generating a Recombinant DNA Molecule
The complementary cohesive ends created by restriction enzymes can anneal, but the relatively weak
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