Oligonucleotide primers

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

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. 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

In all amplification experiments, oligonucleotide primers were used at a final concentration of 1 / M. PCR was performed for 35 cycles (1 min at 94 C- denaturation-, 1 min at 50 C-annealing-, 2min at 72 C-extension-) followed by 5 min at 72 C of final extension. 

The second experiment was carried out with the same RNA isolated from mycelia growing on presence of glucose and apple pectin. cDNAs were obtained from those RNA by reverse trancription and used as template for PCR assays. Specific oligonucleotide primers for PG gene were used in the amplification reaction. The Fig-5 shows the results of this amplification experiment. 

The method of PCR allows selective amplification from a complex genome by enzymatic amplification in vitro. The double-stranded genomic DNA template is denatured by heating, and the temperature is then decreased to allow oligonucleotide primers to hybridize (anneal) to their complementary sequences on opposite strands of the template. The... 

PCR is a technique for in vitro amplification of DNA sequences that involves repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension [29], The amplified products following PCR cycles contain double-stranded DNA fragments of discrete length. These DNAs are copies of the template DNA that are bounded at the 5 -terminus by the oligonucleotide primer for the sequence extension with a heat-resistant DNA polymerase. In quantitative assays of PCR products, therefore, nonspecific products interfere with the assay. 

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

Amplify the insert sequence using suitable oligonucleotide primers. 

The Cadet laboratory has prepared phosphoramidites of stereoisomers for 5, 8-cyclo-dA and 5, 8-cyclo-dG for incorporation into oligonucleotides. Primer extension assays using the mammalian replicative enzyme pol 8 demonstrated that 5, 8-cyclonucleosides block DNA replication in vitro and thus would be highly... 

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