Biotechnology Approaches in the Synthesis of Biopolymers

There are two techniques for polynucleotide synthesis that are applied for the production of medium- to large-sized DNA. PCR is commonly utilized for amplification or modification of medium-sized DNA motifs (150-10,000 base pairs). For larger DNA sequences ( 10,000 base pairs), genetic engineering is preferred. Both require solid phase synthesis of DNA sequences primers for PCR or short DNA pieces for gene assembly in genetic engineering methods. 

PCR reactions utilize certain thermostable DNA polymerases, which can synthesize DNA at 70-80°C and do not denaturate at higher temperature ( 98°C). These polymerases allow for the stepwise heating/cooling cyclic steps that are required for DNA denaturation, primer annealing, and DNA elongation when appropriate primers are present (Fig. 3.50). Briefly, the first step of the cycle is DNA denaturation (94-98°C), which is followed by an 

FIGURE 3.50 Schematic view of PCR cycle (left) and propagation (right). (See insert for color representation of the figure.) 

DNA polymerases utilized in PCR have different synthetic specificity, which is defined by the mutation frequency (about errors per bp). Therefore, sequence verification is commonly needed for PCR because of high mutation probability. Some applications that are not related to the gene or protein expression have a high tolerance for mutations and therefore do not require the expensive sequencing step. 

RNA biosynthesis is similar to DNA synthesis but includes one extra step, which is transcription of DNA into RNA molecule by RNA polymerase enzyme. For replication of natural RNA of interest, a reverse transcription is 

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