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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 ...
Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is a quasilinear whole genome amplification method. Unlike conventional DNA amplification methods that are non-linear or exponential (in each cycle, DNA copied can serve as template for subsequent cycles), MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from ...
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 ...
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.
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.
Following annealing of the primer to the template, DNA replication proceeds to the end of the template. The duplex is denatured and the second primer anneals to the newly formed DNA strand, containing sequence from the first primer. Replication proceeds to produce a strand of the required sequence, containing the mutation.
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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 ...