Double-stranded deoxynucleotide

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. 

HIV-1 RT bound to double-stranded ohgonucleotide template-primers both in the presence and in the absence of a deoxynucleotide triphosphate (dNTP) substrate... 

The polymerase chain reaction. Double-stranded ONA is heated to 9S"C in (he presettce of two short oligonucleotide primer sequences, each of which is complementary to the end of one of the strands. After the DNA denatures, the temperature is lowered and the primer sequences anneal to the strand ends. Raising the temperature in the presence of Tag polymerase, Mg, and a mixture of the four deoxynucleotide triphosphates (cINTP s) effects strand replication, producing two DNA copies. Each further repetition of the sequence again doubles the number of copies. 

Another isothermal amplification technique is strand displacement amplification (SDA). " After heat denaturation of DNA in the presence of four primers, dCTP, dGTP dUTP, and a modified deoxynucleotide (dATPaS), two enzymes are added, an exonuclease-deficient polymerase and a restriction enzyme. The two flanking primers that enter into exponential amplification have a restriction site added to their 5 end and get nicked by the restriction enzyme, allow-ing displacement of strands that can in turn be primed, extended, and nicked. Deoxy-ATPotS is used so that the restriction sites include a hemiphosphorothioate linkage to allow single-strand nicking, instead of cutting through double strands. 

The repair synthesis assay developed by Wood et al. in 1988 [130] is based on a damaged double-stranded plasmid, which is incubated with cell or tissue extract in the presence of normal dNTP and one -P-labeled deoxynucleotide. The incorporation of labeled deoxynucleotides by the repair enzymes in the extract corresponds to the repair capacity. The assay, however, is quite laborious and rarely used for quantitative purposes. 

Every PCR run must include the DNA template, two primers complementary to the target sequence, four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), DNA polymerase, and MgCl2 mixed in the reaction buffer. There are three steps in the PCR cycle (Pig. 2.2). Pirst, the reaction mixture is heated to a high temperature (95°C), which leads to DNA denaturing (i.e., separation of the double-stranded DNA into two... 

Since DNA is double-strand, damage to one strand of the DNA can often be repaired by using the undamaged complementary strand as a template to direct new incorportion of correct deoxynucleotides in place of the removed incorrect ones. 

DNA polymerase catalyzes the synthesis of the complementary strand of DNA in vitro, using single-stranded DNA as a template, provided that both the four deoxynucleotide triphosphate (dNTP) monomers and a primer are present.The primer provides a short stretch of double-stranded DNA by base pairing with its complement on the single-stranded DNA. 

The mode of binding of DNA polymerase to DNA depends upon whether the DNA is single or double stranded. With an unbroken double-stranded DNA, the polymerase binds only at the ends of the molecules. If strands have broken, the enzyme binds wherever there is a nick, provided that nicking yields a free 3 -OH. In contrast, binding with single-stranded DNA may occur at several different sites along the chain. The existence of another binding site for mono- and di-deoxynucleotides has also been reported. 

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