Ac Tg

Figure 7.3. Recognition sites of some common restriction endonucleases.

Bacteriophages (page 74) are viruses that infect bacteria and utilize the host cell's components for their own replication. The bacteriophage genome is, like a plasmid, circular, although many viruses use RNA rather than DNA as their genetic material. If human DNA is inserted into a bacteriophage, the bacteriophage will do the job of introducing it into a bacterium.

Joining Foreign DNAs to a Cloning Vector. Enzymes known as restriction en-donucleases are used to insert foreign DNA into a cloning vector. Each restriction endonu-clease recognizes a particular DNA sequence of (usually) 4 or 6 bp. The enzyme binds to this sequence and then cuts both strands of the double helix. Many restriction endonucle-ases have been isolated from bacteria. The names and recognition sequences of a few of the common ones are shown in Figure 7.3. Restriction endonuclease names are conventionally written in italics because they are derived from the Latin name for the bacterium in which the protein occurs.

Some enzymes such as Bam HI, Eco RI, and Pst I make staggered cuts on each strand. The resultant DNA molecules are said to have sticky ends (Fig. 7.4) because such fragments can associate by complementary base pairing to any other fragment of DNA generated by the same enzyme. Other enzymes such as Sma HI cleave the DNA smoothly to produce a sticky end a blunt end

Figure 7.4. Restriction endonulceases generate two types of cut ends in double-stranded DNA.

blunt ends (Fig. 7.4). DNA fragments produced in this way can be joined to any other blunt-ended fragment.

Figure 7.5 illustrates how human DNA is inserted into a plasmid that contains a Bam H1 restriction endonuclease site. A short length of synthetic DNA (an oligonucleotide) that includes a Bam H1 recognition site is added to each end of the human DNA fragment. Both the human DNA and the cloning vector are cut with Bam H1. The cut ends are now complementary and will anneal together. DNA ligase then catalyzes the formation of phospho-diester links between the vector and the human DNA. The resultant molecule is known as a recombinant plasmid. If our starting material was mRNA from a sample of liver, we would now have a collection of plasmids each carrying a cDNA from one of the genes that was being transcribed in this organ.

Introduction of Recombinant Plasmids into Bacteria. Figure 7.6 summarizes how recombinant plasmids are introduced into bacteria such as Escherichia coli. Bacteria are first treated with concentrated calcium chloride to make the cell wall more permeable to DNA. DNA can now enter these cells, which are said to have been made competent. Cells that take up DNA in this way are said to be transformed. The transformation process is very inefficient, and only a small percentage of cells actually take up the recombinant molecules. This means that it is extremely unlikely that any one bacterium has taken up two plasmids. The presence of an antibiotic resistance gene in the cloning vector makes it possible to select those bacteria that have taken up a molecule of foreign DNA, since only the transformed cells can survive in the presence of the antibiotic. The collection of bacterial colonies produced after this selection process is a clone library. All the cells of a single colony harbor identical recombinant molecules that began as one mRNA molecule in the original cell sample. Other colonies in the same clone library contain plasmids carrying different DNA inserts. Isolating individual bacterial colonies will produce different clones of foreign DNA. In the example we have described, where the starting DNA material used to produce these clones was a population of cDNA molecules, the collection of clones is called a cDNA library.

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