246
DNA Cloning and Characterization
In DNA cloning, recombinant DNA molecules are formed in vitro by inserting DNA fragments into vector DNA molecules. The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules.
Restriction enzymes (endonucleases) typically cut DNA at specific 4–
Two restriction fragments with complementary ends can be joined with DNA ligase to form a recombinant DNA molecule (see Figure 6-12).
E. coli cloning vectors are small circular DNA molecules (plasmids) that include three functional regions: a replication origin, a selectable marker gene, and a site where a DNA fragment can be inserted. Transformed cells carrying a vector grow into colonies on the selection medium (see Figure 6-14).
A genomic library is a set of clones carrying restriction fragments produced by cleavage of the entire genome.
Shuttle vectors that can replicate in both yeast and E. coli can be used to construct a yeast genomic library. Specific genes can be isolated by their ability to complement the corresponding mutant genes in yeast cells (see Figure 6-16).
In cDNA cloning, expressed mRNAs are reverse-
The polymerase chain reaction (PCR) permits exponential amplification of a specific segment of DNA from a single initial template DNA molecule if the sequence flanking the DNA region to be amplified is known (see Figure 6-19).
PCR is a highly versatile method that can be programmed to amplify a specific genomic DNA sequence, a cDNA, or a sequence at the junction between a DNA transposon and flanking chromosomal sequences.
DNA fragments up to about 100 nucleotides long can be sequenced by generating clusters of identical fragments by PCR and imaging fluorescently labeled nucleotide precursors incorporated by DNA polymerase (see Figures 6-21 and 6-22).
Whole genome sequences can be assembled from the sequences of a large number of overlapping clones from a genomic library (see Figure 6-23).