DNA primer

In recent years, automated DNA sequencing machines capable of identifying about 10 bases per day have become commercially available. One clever innovation has been the use of fluorescent dyes of different colors to uniquely label the primer DNA introduced into the four sequencing reactions for example, red for the A reaction, blue for T, green for G, and yellow for C. Then, all four reaction mixtures can be combined and run together on one electrophoretic... 

Experiments were carried out which showed that the ratio of TT/UT (4 8) observed in the acid hydrolysate of a DNA was nearly the same as the ratio (5 2) found in the products of the more gentle enzymatic hydrolysis. Other experiments showed that the photoreactivating enzyme could excise all three dimers equally readily, that all three dimers were implicated in the photoinactivation of primer DNA, and that CT dimers were excised from the DNA of radiation-resistant bacteria. 

RNA polymerase, and the triphosphates of the purine ribonucleosides, uridine, and cytidine, and otherwise the same conditions, will prime the synthesis of RNA. The amounts of synthetic DNA or RNA are many fold greater than the amount of primer DNA the DNA product is nearly the same in most measurable ways as the primer DNA. The efficiency of the DNA in initiating these syntheses is known as the primer activity of the DNA, and can be affected by alterations of the bases which compose the nucleic acid, and by other factors. 

Add primer, DNA polymerase + dATP, dGTP, dCTP, dTTP. 

Many variations of site-directed mutagenesis exist. One can start out with a circular, single-stranded DNA and anneal it to a synthetic primer DNA carrying the desired changes (fig. 27.10). This primer can be extended, and the resulting product can be transfected. Finally, one selects clones of cells containing the plasmid with the desired changes. 

RNA primer DNA polymerase cannot start DNA synthesis without a primer. Even on the... 

Chain termination An incubation mixture is set up containing the single-stranded DNA template, method the primer, DNA polymerase I and all four deoxyribonucleoside triphosphates... 

Chain initiation occurs when a specialized RNA polymerase enzyme called primase makes a short RNA primer. DNA polymerase III extends this RNA primer on both strands. Because DNA polymerase synthesizes DNA only in one direction (5 to 3 ), only one strand is copied in each direction (left and rightward in the next figure). At the end of the initiation process, two replication forks exist, going in opposite directions from the bubble at the origin of replication, as shown in Figure 8-11. 

Two types of primer DNA (Sections 4.2.1. and 4.8.1.) may be employed for the chain-extension reaction. When the desired sequence, of chain length up to —400 nucleotides, is cloned into M13mp2, a flanking primer which hybridizes to a region of the... 

Random primer DNA labeling kit (Boehringer-Mannheim, Indianapolis, IN). 

Label the reaction tubes (600-//1 Eppendorf tubes) and add 23 fil of reaction mix (Step 3), 1 fil diluted template DNA (5-100 ng), and 1 fil diluted primer DNA (5-6 pmol). The advantage of adding the reaction mix first is that the surface tension will help pull down the 1 fil volumes, which would otherwise be hard to pipette into the tubes. If using only one template DNA with a number of primers or one primer DNA with a number of templates, the single primer or template DNA can be combined with the mixture in Step 3 instead of adding it separately in Step 4. 

We have found that 0.02 units//d of Tag polymerase is often sufficient for RAPD reactions using 0.2-0.6 ng/pi of template DNA with 0.24 pmol///l of primer DNA, though the optimal quantity of Tag polymerase may depend on the supplier used. Excessive amounts of Tag polymerase, or in some cases template DNA, can cause the reaction to appear as a smear of amplified DNA rather than as discrete bands.4... 

Figure 3-31. DNA sequencing by the dideoxynudeotide method. A DNA template is hybridized with a primer. DNA polymerase is added plus dATP, dGTP, dCTP, and dTTP. Either the primer or the nucleotides must have a radioactive label, so bands can be visualized on the gel by autoradiography. Samples are placed in each of four tubes and one of the four dideoxyribonudeotides (ddNTPs) is added to each tube to cause random termination of synthesis.

An excess of two short primer DNAs that are complementary to opposite strands of the DNA fragment that is to be amplified, and... 

Figure 10.5 (a) Template/primer sequence contexts used for dynamic l9F-NMR and DSC experiments [79]. (b) Example n + 3 translesion synthesis models of BF complexed with AF-modified template/primer DNA n + 3 dC-match (B-type conformer) and n + 3 dA-mismatch (W-conformer) [56]. The carcinogenic aminofluorene moiety is... 

Pol 1 Filling of gap after removal of RNA primer DNA repair Removal of RNA primer in conjunction with RNAse H 5 to 3 and 3 to 5 ... 

Pol a Replication (in a complex with primase and aids in starting the primer) DNA repair None... 

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