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Figure 13.3 A methylase enzyme adds a methyl group to cytosine, converting it to 5-methylcytosine.
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Sequence recognized 3 G T Py Pu A C C A Pu Py T G 5 3 G A A T T C C T T A A G 3 5 5 G G A T C C C C T A G G 3 5 C T G C A G G A C G T C 3 5 5 3 5 3 5 3 5 C T G C A 3 G 5 3 G T Py C A Pu 3 5 5 3 Pu A C Py T G 3 Blunt ends 5 3 5 Overhang 5 3 5 Overhang 5 3 G A C G T C 3 Overhang 5
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Figure 13.2 Sequences cleaved by various type II restriction endonucleases. Py is any pyrimidine and Pu is any purine. Arrows denote places where endonucleases cleave the DNA. In 1971, K. Danna and D. Nathans showed that a restriction endonuclease would consistently cut DNA into pieces of the same size. This precision and repeatability of enzyme action made enzymes useful for further research. Not all restriction endonucleases make staggered cuts with 3 and 5 overhangs; some produce blunt ends.
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Thirteen
Genomics, Biotechnology, and Recombinant DNA
endonucleases attack in the unmethylated condition are protected when methylated. After host DNA replication, new double helices are hemimethylated; that is, the old strand is methylated but the new one is not. In this con guration, the new strand is quickly methylated ( g. 13.4). Foreign DNA, without methyl groups on either strand, is not methylated. Restriction endonucleases are named after the bacteria from which they were isolated: BamHI from Bacillus amyloliquefaciens, strain H; EcoRI from E. coli, strain RY13; HindII from Haemophilus in uenzae, strain Rd; and BglI from Bacillus globigii. From here on, we will refer to type II restriction endonucleases simply as restriction enzymes. Restriction enzymes cut the DNA in two different ways. For example, HindII cuts the recognition sequence down the middle, leaving blunt ends on the DNA (see g. 13.2). We will discuss how pieces of DNA with blunt ends can be used in cloning.The staggered cuts made, for example, by BamHI leave sticky ends (a 5 overhang) that can reanneal spontaneously as hydrogen bonds form between the complementary bases (see g. 13.2). The ability to reanneal these sticky ends, rst demonstrated by S. Cohen, H. Boyer, and colleagues in 1971, opened up the eld of gene cloning.
we see how a circular DNA molecule cleaved by a speci c restriction enzyme can recircularize if it is cleaved in only one place, or how different molecules with the same free ends can anneal to form hybrid molecules. Only the action of a DNA ligase is needed to make the molecules complete (see chapter 9). One of the pieces of DNA involved in the annealing can be a plasmid, a piece of DNA that can replicate in a cell independently of the cellular chromosome. The recombinant plasmid (fig. 13.6) can be transferred into a cell. (A recombinant plasmid is also known as a hybrid plasmid, hybrid vehicle, hybrid vector, or chimeric plasmid. The latter is after the chimera, a mythological monster with a lion s head, a goat s body, and a serpent s tail.) Many procedures exist that can introduce this recombinant plasmid into a host cell. For example, a bacterial cell can be made permeable to this, or any, plasmid by the addition of a dilute solution of calcium chloride. Once inside the cell, the foreign DNA is replicated each time the plasmid DNA replicates. Note that in the process of inserting a piece of foreign DNA, the restriction site is duplicated, with one copy at either end of the insert. This property makes it easy to remove the cloned insert at some future time, if needed, since restriction sites enclose it ( g. 13.6).
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