taq dna polymerase extension at 3' end with intra loop does not proceed
Answers
Hyone-Myong Eun, in Enzymology Primer for Recombinant DNA Technology, 1996
i.
Primary functions of DNA polymerases.
DNA polymerases are a group of polymerases that catalyze the synthesis of polydeoxyribonucleotides from mono-deoxyribonucleoside triphosphates (dNTPs), performing the most fundamental functions in vivo of DNA replication, repair, and, in some cases, cell differentiation. In fact, different types of DNA polymerases have been found in a single organism, for example, three (DNA Pol I, II, and III) in E. coli or five (DNA Pol α, β, γ, δ, and ɛ) in higher eukaryotes, which are believed to perform a specialized in vivo function(s). With somewhat different complexities from in vivo functions, DNA polymerases can, when given suitable conditions, also perform DNA synthesis in vitro. They require, in addition to dNTPs, an initiating oligonucleotide (or polynucleotide), called a primer, carrying a 3′-end hydroxyl group that can be used as the starting point of chain growth (1, 2). DNA polymerases cannot initiate synthesis de novo from mononucleotides.
A primer can be a short or long piece of DNA or RNA which carries a free 3′-OH group. Primers provide a double-stranded structure to the DNA polymerase by annealing to a complementary region of the DNA or RNA strand called a template. The DNA polymerase moves along the DNA (or RNA) template, extending the primer in the 5′ → 3′ direction according to the Watson–Crick base pairing rule, i.e., A pairs with T (or U) and C pairs with G (see Section II, Chapter 1). The polarity of the newly synthesized chain is opposite (or antiparallel) to that of the template. Incorporation of a noncomplementary nucleotide is considered an “error.” The error frequency (or fidelity) is an important characteristic of a polymerase (see below).
In addition to the major 5′ → 3′-polymerase activity, a DNA polymerase may exhibit several other activities, such as 5′-nuclease, 3′ → 5′-exonuclease, and/or RNase H activities, which are necessary for proper in vivo functions.
The initiation of cellular DNA replication takes place at a single site (e.g., oriC of E. coli) or multiple specific sites (in higher eukaryotes) of DNA called origins of replication (ori). The temporal site of dsDNA where the replication occurs is called a “replication fork.” Because of DNA strand polarity, the bidirectional replication results in two distinct products, “leading” and “lagging” strands, according to the moving direction of the replication fork. The leading strand is synthesized as a single continuous chain, whereas the lagging strand is initially synthesized as small oligonucleotides, called Okazaki fragments, which are then ligated to form a continuous chain. Small RNAs play an important role as natural primers in the synthesis of both the leading strand and, in particular, the lagging strand.