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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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13. Genomics, Biotechnology, and Recombinant DNA
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The McGraw Hill Companies, 2001
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Primer 5 (b) A A A A A A 3 mRNA T T T T T T 5 DNA Reverse transcription 5 (c) 3 RNase H 5 (d) 3 A A A A A A 3 mRNA T T T T T T 5 DNA A A A A A A 3 mRNA T T T T T T 5 DNA (first strand)
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Polymerase I 5 (e) 3 A A A A A A 3 DNA (second strand) T T T T T T 5 DNA
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DNA ligase 5 (f) 3 Figure 13.10 A A A A A A 3 DNA T T T T T T 5 DNA
(a) A messenger RNA, shown in black, begins as a single strand. (b) A poly-T DNA segment (red) is added as primer; it complements the 3 poly-A tail of the eukaryotic messenger RNA. (c) Reverse transcriptase acts on this primed con guration to synthesize a single strand of DNA from the RNA template. (d) The RNA is then nicked randomly by RNase H. (e) The RNA segments are then replaced by DNA (blue) by the action of DNA polymerase I. (f) After DNA ligase treatment, the nal result is double-stranded complementary DNA (cDNA).
the genome. The gene of interest can be found either before or after cloning it, although it is usually done after cloning.
Creating a Genomic Library
When cDNA or synthetic DNA cannot be used for cloning, the total DNA of an organism can be broken into small pieces to isolate the desired gene or DNA fragment. The desired DNA can be isolated either before or after cloning. This DNA is referred to as genomic DNA to differentiate it from cDNA. If the original DNA is isolated before cloning, then only that DNA need be cloned. Alternatively, a shotgun ap-
proach can be used to clone a sample of the entire genome of an organism (in small pieces, of course), creating a genomic library, a set of cloned fragments of the original genome of a species ( g. 13.11). In a genomic library, a desired gene can be located after it is already cloned.
Southern Blotting
When DNA segments are generated randomly, usually by endonuclease digestion, a desired gene must be located. As mentioned, we can look for the gene either before or after it is cloned. We consider rst the procedure for locating a speci c gene in a DNA digest, before the DNA has been cloned.
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Genomic Tools
To locate a speci c gene in the midst of a DNA digest, one must have a speci c probe. Probes are generally nucleic acids with sequences that precisely locate a complementary DNA sequence by hybridization. The probes are labeled so they can be identi ed later with autoradiography or chemiluminescent techniques (techniques in which tags are used that uoresce under ultraviolet or laser light). Thus, if we wish to locate the gene for -globin, we could use radioactively labeled -globin messenger RNA or radioactively labeled cDNA. RNA-DNA or DNA-DNA hybrids would form between the speci c gene
and the radioactive probe. Autoradiography or chemiluminescence would then locate the radioactive probe. Let us assume that we wanted to clone the rabbit -globin gene. First, we would create a restriction digest of rabbit DNA ( g. 13.12). We would then subject this digest to electrophoresis on agarose to separate the various fragments according to size. Agarose is a good medium for separating DNA fragments of a wide variety of sizes.
Begin with genome of organism
Create fragments with blunt ends or restriction-generated sticky ends
Clone in plasmids or phage
Amplify and isolate vector in E. coli Plate out Produce plaques containing genomic library
Creating a genomic library using the shotgun approach in creating inserts. First, the genome is fragmented. The fragments are then cloned randomly in vectors. The collection of these vectors is referred to as a genomic library.
Figure 13.12 Locating the rabbit -globin gene within a DNA digest using the Southern blotting technique. The rabbit DNA (a) is segmented with a restriction endonuclease (b) and then electrophoresed on agarose gels (c). Southern blotting transfers the DNA to nitrocellulose lters (d). Finally, a radioactive probe ( -globin messenger RNA) locates the DNA fragment with the -globin gene after autoradiography (e).
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