Polymerase primer

Unexpectedly low levels of PCR product in positive controls are seen periodically. On different occasions, we identified problems with Taq polymerase, primers, or RNA degradation. Primer concentrations may need to be remeasured, for verification that the amount being added is correct. Unknown differences in quantity of primer will result in problems with the PCR efficiency (13). Degradation of primers or of RNA will result in high background and low PCR product. 

After initial tests, the next step is to optimize steps of the analytical process, including nucleic acid extraction, amplification, detection, and quantification. For amplification procedures, variables to be optimized may include buffer pH, nucleotide concentration, MgCb concentration, type of polymerase, primer concentration, and annealing temperature. 

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. 

The elongation of the primers with the complementary order of nucleotides by the synthesis of the new DNA strand by the enzymatic activity of the taq-polymerase (primer extension)... 

Figure 4 schematically describes the synthesis of a probe. A universal sequencing primer (e.g., 1211, BRL) is annealed to its complementary sequence in the vicinity of the M13 polylinker. Klenow polymerase is then used to elongate the primer and synthesize the complementary strand of the probe insert which will be highly labeled if [a pj dATP of high specific activity is Included in the reaction. Provided that polymerase, primer and cold nucleotides are in excess, primer elongation will start... 

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. 

It should be emphasized that the polymerase chaia reactioa requires specific primers for syathesis therefore, the sequeace flanking the sequeace to be amplified must be known. PGR reactioas are also very susceptible to contamination by other DNA. Precautioas agaiast contamination aeed to be... 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

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... 

The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion. the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

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