DNA Cloning by Recombinant DNA Methods
■ 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- to 8-bp palindromic sequences, producing defined fragments that often have self-complementary single-stranded tails (sticky ends).
■ Two restriction fragments with complementary ends can be joined with DNA ligase to form a recombinant DNA (see Figure 9-11).
■ E. coli cloning vectors are small circular DNA molecules (plasmids) that include three functional regions: an origin of replication, a drug-resistance 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 9-13).
■ Phage cloning vectors are formed by replacing nonessential parts of the \ genome with DNA fragments up to «25 kb in length and packaging the resulting recombinant DNAs with preassembled heads and tails in vitro.
■ In cDNA cloning, expressed mRNAs are reverse-transcribed into complementary DNAs, or cDNAs. By a series of reactions, single-stranded cDNAs are converted into double-stranded DNAs, which can then be ligated into a \ phage vector (see Figure 9-15).
■ A cDNA library is a set of cDNA clones prepared from the mRNAs isolated from a particular type of tissue. A genomic library is a set of clones carrying restriction fragments produced by cleavage of the entire genome.
■ The number of clones in a cDNA or genomic library must be large enough so that all or nearly all of the original nucleotide sequences are present in at least one clone.
■ A particular cloned DNA fragment within a library can be detected by hybridization to a radiolabeled oligonu-cleotide whose sequence is complementary to a portion of the fragment (see Figures 9-16 and 9-17).
■ Shuttle vectors that 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 9-20).
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