1. DNA sequences can be cloned using reverse transcriptase, which copies mRNA into DNA to make a hybrid mRNA:DNA double-stranded molecule. The mRNA strand is then converted into DNA by the enzymes ribonuclease H and DNA polymerase. The new double-stranded DNA molecule is called complementary DNA (cDNA).
2. Restriction endonucleases cut DNA at specific sequences. DNA molecules cut with the same enzyme can be joined together. To clone a cDNA, it is joined to a cloning vector—a plasmid or a bacteriophage. Genomic DNA clones are made by joining fragments of chromosomal DNA to a cloning vector. When the foreign DNA fragment has been inserted into the cloning vector, a recombinant molecule is formed.
3. Recombinant DNA molecules are introduced into bacterial cells by the process of transformation. This produces a collection of bacteria (a library) each of which contains a different DNA molecule. The DNA molecule of interest is then selected from the library using either an antibody or a nucleic acid probe.
4. There are many important medical, forensic and industrial uses for DNA clones. These include:
• Determination of the base sequence of the cloned DNA fragment.
• In situ hybridization to detect specific cells making RNA complementary to the clone.
• Southern blotting and genetic fingerprinting to analyze an individual's DNA pattern.
• Synthesis of mammalian proteins in bacteria or eukaryotic cells.
• Changing the DNA sequence to produce a new protein.
• Generation of fluorescent protein chimeras for subsequent microscopy on live cells.
• The polymerase chain reaction, which lets us produce many copies of a DNA molecule in a test tube.
• Production of transgenic animals to study gene function.
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