Hybridization oligonucleotide primers

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... 

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. 

The polymerase chain reaction utilizes a thermostable DNA polymerase to amplify DNA through a series of temperature cycle steps. The key to the specificity of the reaction is the selection of oligonucleotide primers that hybridize to the opposite strands of the DNA being tested, about 400-2000 bp apart. If the sequence of the primers is unique within the genome, and the primers hybridize to the target DNA at a high enough temperature to avoid close matches (various... 

Synthesis of a specific DNA segment via the PCR is directed by two oligonucleotide primers. Multiple rounds of extension from the primers amplify the target sequence exponentially, with termini defined by the 5 ends of the two primers. This process allows the determination of sequences from a very small number of target molecules. Unique sequences can be amplified from a complex mixture of nucleic acids because of the high specificity of the primer-template hybridization. 

A single DNA molecule can be amplified millions of times by replicating a three-step cycle. In the first step the dsDNA sample is denatured by heating to 95°C. In step 2 the temperature is quickly lowered to 50°C and an oligonucleotide primer is added. The primer hybridizes to complementary sequences on the ends of the two strands. During step 3, DNA synthesis occurs as the temperature is raised to 70°C, the optimal temperature of Taq polymerase. The cycle is then repeated with both old and new strands serving as templates. 

PCR reaction (25 yL final volume) contained Taq polymerase, 100 pmoles of the oligonucleotides primers, 2500 pmoles dNTP, T. aestivum genomic DNA (20 to 100 ng). 25 to 30 cycles were performed. A 1 min denaturation step at 94°C was followed by a hybridization step at 60 or 62°C for 0.5 to 2 min, followed by a 1.5 min elongation step at 72°C. The experiment was terminated by a 4 min step at 72°C. The PCR fragments were purified using Wizard PCR prep kit, either directly or after electrophoresis separation, and were cloned in the PGEM-T vector. The plasmidic DNA were first restriction analyzed, and then sequenced by MWG Biotech (Germany). 

Enzymatic reactions commonly used to synthesize double-stranded cDNA from mRNA and oligonucleotide primers such as oligo d(T). The enzyme reverse transcriptase is an RNA-directed DNA polymerase first discovered in eukaryotic retroviruses. The conversion of RNA to DNA in step 2 is accomplished by combining the endonucleolytic activity of E. coli RNase H to create nicks in the RNA-DNA hybrid with the DNA polymerizing activity of E. coli DNA pol I, which uses the residual RNA as a primer and the first-strand cDNA as a template. E. coli DNA... 

Figure 5.7-7. Schematic overview of different SNP genotyping approaches. (A) Allelic hybridization. (B) Primer extension. (C) Oligonucleotide ligation. (D) Invasive cleavage. (This figure is available in full color at ftp //ftp.wiley.com/public/sci tech med/ pharmaceutical biotech/.)...

The cumbersome hybridization procedures led to the use of PCR-based identification methods. Short, synthetic oligonucleotide primers allowed the amplification of the desired genomic regions of the target species. The ampMcation products were then analyzed using many different methods. 

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