Template-directed primer extension

DNA-directed DNA polymerases [EC 2.7.7.7], also called DNA nucleotidyltransferases (DNA-directed), are enzymes that catalyze the DNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . This enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. RNA-directed DNA polymerases [EC 2.7.7.49], also referred to as reverse transcriptases, DNA nucleotidyltransferases (RNA-directed), and revertases, are enzymes that catalyze the RNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . As was the case above, this enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. 

This enzyme [EC 2.7.7.49], also known as RNA-directed DNA polymerase, DNA nucleotidyltransferase (RNA-directed), and revertase, catalyzes the RNA-template-directed extension of the 3 -end of a DNA strand by one deoxynucleotide at a time n deoxynucleoside triphosphate to produce n pyrophosphate (or, diphosphate) and DNA . The enzyme cannot initiate a DNA chain de novo and requires a DNA or RNA primer. See also Viral Polymerases... 

Fig. 13. Schematic of the single-base extension assay applied to Tag probe arrays. Regions containing known SNP sites (A or G in this example) are first amplified by PCR. The PCR product serves as the template for an extension reaction from a chimeric primer consisting of a 5 tag sequence and a 3 sequence that abuts the polymorphic site. The two dideoxy-NTPs that could be incorporated are labeled with different flurophors in this example, ddUTP is incorporated in the case of the A allele, and ddCTP for the G allele. Multiple SBE reactions can be done in a single tube. The tag sequence, unique for each SNP, directs the extension products to a particular address on the Tag probe array. The proportion of a fluorophor at an address reflects the abundance of the corresponding allele in the original DNA. (Reprinted with permission from [45])...

DASH, dynamic allele-specific hybridization AS-PCR, allele-specific polymerase chain reaction AS-PE, allele-specific primer extension APEX, arrayed primer extension FP-TDI, fluorescence polarization template directed dye terminator incorporation MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry OLA, oligonucleotide ligation assay RCA, ... 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

Due to all these constrains, the DNA replication becomes a complicated process (see schematics in Fig. 4). The process begins when a special type of RNA polymerase called primase synthesizes short RNA primers, which then extended by DNA polymerase. On one of the two strands, where primer extension proceeds in the 5 3 direction, the synthesis is straightforward (it is called the leading strand, see Fig. 4). But how can primer be extended on the opposite, so-called lagging, strand as a template It is done in a very nonelegant way short pieces of DNA are synthesizes in the 5 3 direction, i.e. 

All DNA polymerases catalyze the template-directed incorporation of deoxy-ribonucleotides (dNTPs) into DNA by addition at the 3 OH termini of primer strands. Primer extension proceeds exclusively in the 5 to 3 direction, according to the reaction ... 

As an alternative to this approach, one can directly measure dNTP analog incorporation and extension using a gel-based assay and a DNA template consisting ofaS - P -labeled primer, annealed to a single-stranded template (as an example, see ref. 44). Primer extension in the presence of three dNTPs produces a block that can be overcome with the addition of incorporable analogs with extensible 3 -OH termini. 

In certain cases, the last two criteria may be simultaneously met if the same polymerase can amplify unmodified DNA into modified DNA. Although direct exponential amplification of modified DNA into more modified DNA affords greater convenience, such was initially thought to be a necessary requirement for selections. However, the simultaneous satisfaction of both criteria 2 and 3 is not a requirement for use in selections for the same reason that early aptamers were composed of RNA which necessitated both RT (criterion 2) and amplification (criterion 3). In fact, a primer extension reaction along an unmodified template to produce modified DNA without exponential amplification, followed by selection and subsequent recopying into unmodified DNA prior to exponential amplification, may actually eliminate extraneous selection pressures that are imposed by demanding that both strands (selected and nonselected) be composed of modifications, as would be the case in PCR. 

Reverse transcriptase (RT) plays a critical role in the early steps of the life of human immunodeficiency virus (HIV) (304), and for over a decade has been one of the major targets of AIDS therapy. Polycitone A (280) was found to be a potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases (305). Polycitone A exhibited potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. It inhibits retroviral reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV), murine leukemia virus (MLV) and mouse mammary tumor virus (MMTV)] as efficiently as cellular DNA polymerases of both DNA polymerases a and p and the prokaryotic Klenow fragment of Escherichia coli DNA polymerase I. The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A (280) have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension, as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the... 

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

The primers used are restriction fragments which originated from within the duplex DNA to be sequenced. In the annealing reaction, to produce the primer-template complex, only one of the primer strands is hybridized to its complementary sequence in the template since the other potential template is destroyed by the Exo III treatment. The result is the formation of a primer-template which can be extended by DNA polymerase in a direction opposite to that of the Exonuclease III attack. This is shown in Fig. 3.15. Two inherent features of the method which can cause problems are also made clear in Fig. 3.15. In the first place priming and chain extension can also occur on the 3 -ends of both template strands when sufficient complementarity remains between the 3 -ends of the template strands to form a base-paired structure. Secondly, a primer which originates from near the centre of the duplex DNA... 

---