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FIGURE 20-10 Protonatedphospho-ramidile. As ihown, the 5'-hydroxyl group is blocked by the addition of 9 cimethoxyltniyl proteding group.
addition are chemically protected molecules called phosphoamidines (Figure 20-10). Grawlh of the DNA chain is by addition to the 5F end of the molecule, in contrast to the direction of chain growth used by DNA polymerases.
Chemical synthesis of DNA molecules up to 30 bases long is efficient and accurate, and takes only a few hours. It is a routine procedure: a researcher can simply program a DNA synthesizer to make any desired sequence by typing the base sequence into a computer controlling the machine. Rut as the synthetic molecules get longer, the final product is less uniform due to the inherent failures that occur during any cycle of the process. Thus, molecules over 100 nucleotides or so are difficult to synthesize in the quantity and with the accuracy desirable for most molecular analysis.
The rather short DNA molecules that can readily be made, however, are well suited for many purposes. For example, a custom-designed oligonucleotide harboring a mismatch to a segment of cloned DNA can be used to create a directed mutation in that cloned DNA. This method, called site-directed mutagenesis is performed as follows. The oligonucleotide is hybridised to the cloned fragment, and used to prime DNA synthesis with the cloned DNA as template. In this way, a double-stranded molecule with one mismatch is made. The two strands are then separated and that with the desired mismatch amplified further.
Custom-designed oligonucleotides can be used in this manner to introduce restriction sites into cloned DNAs which are then used to create fusions between a coding sequence and another coding sequence or a promoter or ribosome binding site. As another example, synthetic oligonucleotides that have been labeled fluorescently or radioactively can be used as probes in hybridization experiments. Moreover, custom-designed oligonucleotides are critical in the polymerase chain reaction, which we describe next, and are an indispensable feature of the DNA sequencing strategies that we describe below. Therefore, a common feature in designing experiments to construct new molecular clones of genes to detect specific DNAs, to amplify DNAs, and to sequence DNAs is to design and have synthesized a short synthetic DNA oligonucleotide of desired sequence.
The Polymerase Chain Reaction (PCR) Amplifies DNAs by Repeated Rounds of DNA Replication in Vitro
A powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within a host cell, is the polymerase chain reaction (PCR). This procedure is carried out entirely biochemically, that is, in vitro. PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucieotide substrates on a single-stranded DNA template. As we saw in Chapter B, DNA polymerase synthesizes DNA in a 5F to 3' direction and can add nucleotides to the 3' end of a custom-designed oligonucleotide. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate it in a 5' to 3' direction to generate an extended region of double-stranded DNA.
How is this enzyme and reaction exploited to amplify specific DNA sequences? Two synthelic, single-stranded oligonucleotides are synthesized. One is complementary in sequence to the 5' end of one si rand of the DNA to be amplified, the other complementary to the 5' end of the other strand (Figure 20-11). The DNA to be amplified is then denatured and the oligonucleotides annealed to their target sequences. At this nimiiiiimiiniiuiiiiinmmmiiiiiiiiii n i m n 11111 n 11 n 11 i i i i i mil
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