2. DNA polymerase then incorporates a dNMP onto the 3" end of the primer and initiates lagging strand synthesis. The polymerase extends the primer for about 1,000 nucleotides until it comes in contact with the 5' end of the preceding primer. These short segments of RNA/DNA are known as Okazaki fragments.
Many RTs are available from commercial suppliers. and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. lacks 3´ → 5´ exonuclease activity. is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
Glen Research is delighted to introduce a GalNAc modification strategy using a monomeric GalNAc support and the equivalent GalNAc phosphoramidite. Our experimental work has shown that these products are fully compatible with regular oligonucleotide synthesis and deprotection. Oligonucleotides containing GalNAc can be deprotected using standard procedures during which the acetyl protecting groups on the GalNAc group are removed. Glen Research offers these GalNAc C3 products under an agreement with AM Chemicals LLC.
Origins: Origins are unique DNA sequences that are recognized by a protein that builds the replisome. Origins have been found in bacterial, plasmid, viral, yeast and mitochondrial DNA and have recently been discovered in mammalian DNA. Specific origins are used for initiating DNA replication in humans. Most origins have a site that is recognized and bound by an origin-binding protein. When the origin-binding protein binds to the origin the A + T rich sequence becomes partially denatured allowing other replication factors known as cis-acting factors to bind and initiate DNA replication.
Of the three phosphorylation methods described above, only the phosphotriester approach (Fig. 1a) is really suitable for the synthesis of DNA sequences in solution. This method, which was developed largely in the 1970s, is very versatile and is particularly suitable for the coupling of oligonucleotide blocks (i.e., the addition of two or more nucleotide residues at a time) as well as for stepwise synthesis. Phosphotriester block coupling was a key feature of the original synthesis of the human insulin gene (12). Although the methodology has been refined (13) since then, the development of automated solid-phase synthesis (see above) in the 1980s provided a much faster and less labor-intensive method for the preparation of the very small (usually milligram or even smaller) quantities of synthetic DNA sequences that are generally required in molecular biology. Solution-phase synthesis is much more laborious in that it is normally advisable to purify the products by chromatography after each coupling step. Although such purification processes need not necessarily amount to much more than filtration through a bed of silica gel, they are time consuming. Furthermore, solution-phase synthesis has not yet been automated. It is, nevertheless, not at all unlikely that solution-phase synthesis will become the method of choice if really large (i.e., multikilogram to tonne) quantities of moderately sized (containing ca. 20 nucleotide residues) DNA sequences or their analogs are required in anti-sense or antigene chemotherapy. Automated solid-phase synthesis has recently been scaled-up to the multigram level (14) in order to provide sufficient material for clinical trials. However, if such clinical trials are successful and very much larger quantities of pure DNA sequences and their analogs are required for drug purposes, further substantial scaling-up of solid-phase synthesis may not prove to be a practical proposition. It is quite likely that the solution-phase synthesis or perhaps a combination of solution-phase and solid-phase synthesis might lend itself much more readily to scaling-up. The phosphotriester approach has the further advantage that the fully-protected intermediates obtained are soluble in organic solvents and may, therefore, be purified by conventional chromatographic techniques, and, after all of the protecting groups have been removed, the unprotected DNA sequences obtained may, if necessary, be further purified in the same way as material that has been prepared on a solid support.
DNA synthesis requires a primer usually made of RNA. A primase synthesizes the ribonucleotide primer ranging from 4 to 12 nucleotides in length. DNA polymerase then incorporates a dNMP onto the 3' end of the primer initiating leading strand synthesis. Only one primer is required for the initiation and propagation of leading strand synthesis.
Notice that the top strand must be discontinuously replicated in short stretches thus the replication of both parental strands is a semidiscontinuous process. The strand that is continuously synthesized is called the leading strand while the strand that is discontinuously synthesized is called the lagging strand.
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
Since all known DNA polymerases can synthesize only in a 5' to 3' direction a problem arises in trying to replicate the two strands of DNA at the fork.
Relatively high molecular weight DNA sequences have been prepared successfully by the phosphotriester approach in solution by following essentially the procedure indicated in outline in Figure 1a. However, solution-phase synthesis is relatively laborious in that chromatographic purification steps are usually necessary after each coupling step. Nevertheless, if a very large quantity of a specific sequence is required (see text below), solution-phase synthesis may very well prove to be the method of choice. If, on the other hand, relatively small (i.e., milligram to gram) quantities of material are required for biological or biophysical studies, there is little doubt that solid-phase synthesis is to be preferred. While all three of the above phosphorylation methods (Fig. 1) have been used in solid-phase synthesis, the phosphoramidite approach (9) has emerged as the method of choice. This is mainly because its use leads to high coupling efficiencies and no significant side reactions. Furthermore, most commercial automatic synthesizers have been designed specifically to accommodate phosphoramidite chemistry. The main advantages of solid-phase synthesis, particularly by the phosphoramidite approach, are: (1) that it is very rapid and a DNA sequence containing, say, 50 nucleotide residues can easily be assembled and unblocked within one day; (2) only one purification step is required at the end of a synthesis as the growing DNA sequence is attached to a solid support (such as controlled pore glass [CPG] or polystyrene), and the excesses of all reagents are washed away; (3) all chemical reactions can be made to proceed in very high yield by using large excesses of reagents; and (4) the whole process may be fully automated in a DNA synthesizer. Solid-phase DNA synthesis has been developed to such an extent that the whole process can be carried out by a competent technician with no specialist knowledge of nucleotide chemistry. Automatic synthesizers, some of which are capable of assembling several different specific DNA sequences simultaneously, are readily available, and all the necessary building blocks [particularly phosphoramidites 17] and other reagents and solvents may be purchased in containers that are designed to be attached directly to the synthesizer.
The major catalytic step of DNA synthesis is shown below. Notice that DNA synthesis always occurs in a 5' to 3' direction and that the incoming nucleotide first base pairs with the template and is then linked to the nucleotide on the primer.
Newsday NODS OF APPROVAL A single breakout season can earn a major league baseball player the accolade of his own bobblehead doll. Scientists have a tougher time, though. Nobel laureate James Watson had to wait 50 years to earn head-nodding credibility; he joins a small pantheon of scientists so honored. Francis Crick, Watson's colleague in the discovery of DNA's structure, hasn't made the cut, but Albert Einstein has. And for the psychiatry world, Sigmund Freud nods approvingly. ($21.95; www