Primer terminates

Figure 10.3. Mass array, (a) Primer binding (b) primer extension enzyme, ddATP and dCTP/ dGTP/dTTP addition (c) primer terminates (d) primer extension products ready for MALDI-MS (e) MS spectrum of primer extension products. Each addition of a nucleotide to the primer extension product increases the mass by 289 to 329 Da, depending on the nucleotide added. The mass difference is easily resolved by MALDI-TOF, which has the ability to detect differences as small as 3 Da. Printed by kind permission of Sequenom. (See color insert.)...

Topal, M. D., Di Guiseppi, S. R., and Sinha, N. K. (1980). Molecular basis for substitution mutations Effect of primer terminal and template residues on nucleotide selection by phage T4 DNA polymerase in vitro. J. Biol. Chem. 255, 11717-11724. 

We focus on position 75 to limit PyC incorporation to one well-defined location. In certain cases, it may be beneficial to incorporate PyC to both positions C74 and C75 in order to enhance the fluorescence emission signal of the probe. This is achieved by using a tRNA primer terminated at position 73 and by extending the primer with PyCTP and ATP as the nucleotide substrates. In the absence of normal CTP, complete extension of the primer from position 74 to 76, as visualized by denaturing gel analysis (e.g., Fig. 4.3C), is an indication that PyC has been incorporated at both positions. However, due to the rapid reaction of the CCA enzyme to synthesize consecutive C74 and C75 (Dupasquier et al., 2008), it would be difficult to incorporate PyC to just position 74. 

Although the number and location of mismatched bases will affect PCR amplification, mismatches at the 3 terminus are expected to have the greatest effect on the PCR. Kwok et al.9 reported on the effects of 3 -termi-nal mismatches on amplification of HIV sequence. In their system, A-G, G-A, C-C and A-A mismatches were detrimental while all other mismatches had minimal effect. Most notably, oligonucleotides with a 3 -ter-minal T served efficiently as primers even when mismatched with T, C, or G. The concentration of deoxynucleoside triphosphates (dNTPs) in the reaction mix also affects Taq polymerase extension. For example, whereas most 3 -terminal mismatches with the exception of A-G, G-A, C-C, and A-A amplified efficiently in the presence of 800 fiM dNTPs, only T-G and G-T mismatches and perfectly matched sequences amplified in the presence of 6 fiM dNTPs. Thus, 3 -terminal mismatches can be more efficiently extended if the primers terminate in a T and if amplifications are carried out at higher dNTP concentrations. 

Figure 4. Genotyping ofHLA-DQaPCR Product, Type 1.1. Seven primers, comprising four different masses containing 5-mass shifting labels (dB)2 and (dC3) (where n=2 or 3), were selected to type HLA PCR product, each primer terminating one base upstream from the site to be typed. Primer PI did not overlap any of these additional polymorphic sites in the target DNA, but primers P2, P5, and P8 did overlap an additional polymorphism. Primers P2, P5, and P8 ere synthesised as pairs of the same sequence, except for two bases indicated by underlining, one at the 5 end and another at the polymorphic position. An additional terminal 5 base was selected and added to each oligonucleotide sequence in the pair, so that each primer in the pair was of the same base composition and mass.

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.

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. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

A structural feature of the T7 DNA which is important in DNA replication is that there is a direct terminal repeat of 160 base pairs at the ends of the molecule. In order to replicate DNA near the 5 terminus, RNA primer molecules have to be removed before replication is complete. There is thus an unreplicated portion of the T7 DNA at the 5 terminus of each strand. The opposite single 3 strands on two separate DNA molecules, being complementary, can pair with these 5 strands, forming a DNA molecule twice as long as the original T7 DNA. The unreplicated portions of this end-to-end bimolecular structure are then completed through the action of... 

Figure 3-1 A method to GFP epitope tag a gene of interest. (A) Diagram representing the C and N terminal cassettes. The upstream and downstream primers representing 55 nucleotides upstream and downstream (but not including the stop codon) are depicted by I H and respectively, and represent the amplification sequence com-...

Degradation of 5 -terminal sequences in luciferase mRNA containing 60-nt poly(A) tract and capped with the indicated anti reverse cap analogs. The mRNAs half-lives were determined by real time PCR with primers directed against the 5 -end of luciferase mRNA as described in the text. 

This method was developed by Sanger and his colleagues and is also referred to as the chain termination method. The procedure requires a single-stranded DNA template and a short primer complementary to the 3 end of the region of DNA to be sequenced. A complementary copy of the DNA strand is produced... 

Terminal deoxynucleotidyl transferase normally adds homopolydeoxynucleotide tails to single-stranded DNA primers in the presence of a deoxynucleoside triphosphate and magnesium. If cobalt is used instead, not only does double-stranded DNA become an acceptable substrate, but ribonucleotides or homopolymer deoxyribo-nucleotide tracts may be added to all forms of duplex DNA at their 3 -ends, regardless of whether these are staggered or even.162 This allows terminal labelling for sequence analysis at the cleavage sites of restriction endonucleases,162- 183 or tail formation for in vitro studies on recombinant DNA.162... 

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