Single-stranded DNA template

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... 

DNA polymerase III is the main DNA replicating enzyme and consequently is involved in duplication of both the leading and the lagging strands. Besides the deoxyribonu-cleoside triphosphates as substrates, it requires a single-stranded DNA template and an RNA primer. The RNA primer is synthesised according to the DNA sequence at each replication fork. It is DNA polymerase I that is... 

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... 

The single-stranded DNA template and primer are denatured by boiling and then annealed. 

Transcription is one of two processes that allow a cell to synthesize the proteins encoded in its DNA. In the first process, the genes contained on the chromosome are transcribed into molecules of RNA. In the second process, these RNA messages are translated by the ribosomes and transfer RNAs (tRNAs) to produce proteins with specific amino acid sequences. RNA polymerase is the enzyme that synthesizes RNA. This enzyme will read a single-stranded DNA template in the 3 to 5 direction and construct an RNA molecule in the 5 to 3 direction that is complementary to it (Fig. 22-1). In some respects, RNA synthesis is similar to DNA synthesis both molecules are synthesized in the 5 to 3 direction, both of the enzymes involved require a template DNA strand to direct the synthesis, and both of the enzymes involved use nucleoside triphosphates as the basic monomeric units in the synthesis reaction. 

It unwinds a short stretch of double-helical DNA to produce a single-stranded DNA template from which it takes instructions. 

DNA polymerase An enzyme that catalyzes the synthesis of new complementary DNA molecules from single-stranded DNA templates and primers. Different DNA polymerases are responsible for replication and repair of DNA, and they extend the chain by adding nucleotides to the 3 end of the growing DNA catalyzes the formation of covalent bonds between the 3 end of a new DNA fragment and the 5 end of the growing strand. Used in DNA fingerprinting for genealogical studies and forensics. 

DNA polymerases add nucleotides to the 3 end of a polynucleotide chain. The polymerase catalyzes the nucleophilic attack by the 3 -hydroxyl-group terminus of the polynucleotide chain on the a. phosphoryl group of the nucleoside triphosphate to be added (see Figure 4.22). To initiate this reaction, DNA polymerases require a primer with a free 3 -hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free single-stranded DNA template. RNA polymerase, in contrast, can initiate RNA synthesis without a primer (p. 827),... 

Helicases as motors, l lelicases such as RcrA (p. 797) can use single-stranded L9NA as tracks. In each cycle, the helicase moves one base in the. V. 5 direction. Given that PerA can hydrolyze ATP at a rate of 50 molecules per second in the presence of a single-stranded DNA template, calculate the velocity of the helicase in micrometers per second. How does this velocity compare with that of kinesin ... 

If DNA polymerases are added to a single-stranded DNA template with all the deoxynucleotide triphosphates necessary to make a strand of DNA, no reaction occurs. It was discovered that DNA polymerases cannot catalyze de novo synthesis. All three enzymes require the presence of a primer, a short oligonucleotide strand to which the growing polynucleotide chain is covalently attached in the early stages of replication. In essence, DNA polymerases must have a nucleotide with a free 3 -hydroxyl already in place so that they can add the first nucleotide as part of the growing chain. In natural replication, this primer is RNA. 

First, a single stranded DNA template, usually PCR amplified and purified by chromatography, gel filtration or electrophoretic techniques, is immobilised onto a surface. A suitable primer is then hybridised to this single strand (Fig. 5.26). This ensemble is incubated with four enzymes (DNA polymerase, ATP sulfyrase, luciferase and apyrase) and two substrates (adenosine 5 phosphosulfate (APS) and luciferin). 

Fig. 5.26. A single stranded DNA template is immoblised onto a surface and hybridised with a suitable primer.

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