DNA polymerase primer

Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996) Modulation of non-templated nucleotide addition by Taq DNA polymerase primer modifications that facilitate genotyping. Biotechniques 20, 1004-1006, 1008-1010. 

Each primer is a synthetic oligonucleotide of about 20 bases prepared so that then-sequences are complementary to the (previously determined) sequences that flank the tar get regions on opposite strands Thus one primer is annealed to one strand the other to the other strand The 3 hydroxyl end of each primer points toward the target region The stage is now set for DNA synthesis to proceed from the 3 end of each primer [Figure 28 14(c )] The solution contains a DNA polymerase and Mg " m addition to the... 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

The incorporation of acyclovir triphosphate into calf thymus DNA primer template has been shown to be much more rapid and extensive with HSV-1 DNA polymerase than with vero cell DNA polymerase a. This incorporation of acyclovir ceased after 15 min since the template is chain terminated by the acyclovir incorporation, as there is no 3 -hydroxyl group on which to continue elongation. The viral DNA polymerase is also inactivated by tight binding to the terminated template. 

DNA polymerase copies first-strand cDNA using RNA segments as primer... 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Figure 40-7. The polymerase chain reaction is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated several times, giving an amplified product of defined length and sequence. Note that the two primers are present in excess.

Elongation step. The third step is sttand elongation, where the DNA polymerase synthesizes new DNA strands starting at the primer sequences. Under optimum conditions, approximately 60 bp are synthesized per second. Typically, elongation takes place at about 72 °C. 

Saiki, R. K., etal. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-491. 

DNA replication differs between the leading strand and the lagging strand of the DNA double helix. In cells, replication Af the lagging strand involves the formation of short RNA primers by action of an enzyme called RNA primase (or primase for short). Such RNA primers are made at intervals on the lagging strand and are then removed and replaced with DNA by DNA polymerase. 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

Under certain circumstances DNA has both primer and template activities. For example, the addition of mononucleotides is to the 3 end of the growing DNA primer. This presents a problem with regard to how the other strand is synthesized. Biochemists have looked hard but unsuccessfully for an enzyme that can add deoxyribonucleotides onto the 5 end of DNA primers. Such a primer should contain a triphosphate on the hydroxyl group of the 5 end. Although a very active 5 -exonuclease, actually part of DNA polymerase I, has made the search for such an activated 5 end extremely difficult, investigators conclude that a polymerase able to use such a primer probably does not exist. On the contrary, good evidence suggests that the synthesis of both strands is by the known DNA poly-merases. 

All DNA polymerases add mononucleotides to the 3 end of an existing primer. Consequently a special primer is needed for DNA to replicate in its entirety. RNA polymerases can initiate polymer synthesis without a primer thus short RNA primers are used to initiate DNA synthesis. 

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