Characterizing and Verifying Cloned DNA
It is necessary to clone DNA prior to its molecular analysis and subsequent manipulation and application. Once cloned, its characteristics such as fragment size, restriction sites, nucleotide sequence, and subsequence identification (biologically significant sequences) are forthcoming.
Clones are usually between 7–20 Kb, but the targeted sequence (i.e., the gene) is usually only a portion of that fragment. Gross characteristics, such as fragment size and restriction endonuclease sites, can be deduced by combining restriction digestion with agarose gel electrophoresis (Chapter 9) in a technique called restriction mapping. However, restriction mapping does not locate the target sequence. Combining restriction mapping with Southern blotting will determine the location of the target sequence on the clone. Determining the size of the gene (located within the fragment), the precise location of restriction sites, and the identification of subsequences (e.g., start codons, promoters, introns), require detailed data which is obtained by DNA sequencing. This technique combines DNA polymerization (similar to that used in a random probe synthesis) with Polyacrylamide gel electrophoresis.
In the previous laboratory experiments, you performed the steps needed to isolate DNA from yeast, fragment it, ligate the fragments to a vector, insert the recombinant molecules into E. coli, and then screen those transformed E. coli to locate a clone with the MELI gene. The next logical step in this cloning scheme is to characterize the isolated clone. The following laboratory exercises will focus on restriction mapping, Southern blotting, and techniques used for DNA sequencing. The analysis of the DNA sequence will be examined in Chapter 13.
KeywordsTermination Product Restriction Endonuclease Site PstI Site Base Fragment Prehybridization Solution
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- Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning: A Laboratory Manual Plainview, NY: Cold Spring Harbor Laboratory PressGoogle Scholar