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Recombinant DNA (genetic engineering) techniques manipulate DNA fragments
and splice them into combinations that do not occur naturally. 1. Excision of DNA sequences: Exonucleases attack the ends of DNA molecules; endonucleases attack interior DNA sequences. Of these, restriction endonucleases (restriction enzymes such as EcoR I, BamH I, Hind III, Bal I) attack only particular sequences within DNA molecules (usually palindromes, sequences that read the same in both directions). Most restriction endonucleases cut the two DNA strands in slightly different locations, leaving cohesive ("sticky") ends that can join with one another or with other cuts made by the same enzyme. Blunt or flush ends are less useful. 2. Insertion of DNA sequences: DNA sequences with "sticky" ends will join by themselves if they were cut with the same endonuclease and thus have matching ends. "Sticky" ends can join in any order, so there must be some way of selecting only the desired sequences. The enzyme DNA polymerase is used to fill gaps in the sequence, then the enzyme DNA ligase seals the joined molecules. 3. DNA vectors (cloning vehicles): These are DNA sequences that replicate easily. Bacterial plasmids and some viruses (like M13) are naturally occurring DNA vectors. Most DNA vectors are circularly closed, not linear. Ethidium bromide, which binds more easily to linear DNA, can be used to isolate vectors. 4. Cloning of DNA sequences: A DNA probe (a vector bearing an artificially inserted sequence) is inserted into a cell, and the cell is allowed to replicate. The vector must contain genetic traits that permit selection of those cells that have incorporated the vector. Applications of genetic engineering:
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