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Primer extension offers an alternative to a nuclease protection assay (S1 nuclease mapping) for quantifying and mapping RNA transcripts. The hybridization probe for primer extension is a synthesized oligonucleotide, whereas S1 mapping requires isolation of a DNA fragment. Both methods provide information where a mRNA starts and provide an ...
The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur.
These tools are used to optimize the design of primers for target DNA or cDNA sequences. Primer optimization has two goals: efficiency and selectivity. Efficiency involves taking into account such factors as GC-content, efficiency of binding, complementarity, secondary structure , and annealing and melting point (Tm) .
DNADynamo is a commercial DNA sequence analysis software package produced by Blue Tractor Software Ltd [1] that runs on Microsoft Windows, Mac OS X and Linux [2] It is used by molecular biologists [3] to analyze DNA and Protein sequences. A free demo is available from the software developers website.
The strand displacement generates a newly synthesized single-stranded DNA template for more primers to anneal. Further primer annealing and strand displacement on the newly synthesized template results in a hyper-branched DNA network. The sequence debranching during amplification results in a high yield of the products.
Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis. Alternatively, if amplicon sizes overlap, the different amplicons may be differentiated and ...
Reverse transcriptase again synthesizes another DNA strand from the attached primer resulting in double stranded DNA. T7 RNA polymerase binds to the promoter region on the double strand. Since T7 RNA polymerase can only transcribe in the 3' to 5' direction [15] the sense DNA is transcribed and an anti-sense RNA is produced. This is repeated ...
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted.