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Template-switching polymerase chain reaction (TS-PCR) is a method of reverse transcription and polymerase chain reaction (PCR) amplification that relies on a natural PCR primer sequence at the polyadenylation site, also known as the poly(A) tail, and adds a second primer through the activity of murine leukemia virus reverse transcriptase. [1]
OLIGO Primer Analysis Software is a software for DNA primer design. [ 1 ] [ 2 ] The first paper describing this software was published in 1989. [ 3 ] The program is a real time PCR primer and probe search and analysis tool.
A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase (responsible for DNA replication) enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. [1]
During sequencing, each base in the template is sequenced twice, and the resulting data are decoded according to this scheme. SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a next-generation DNA sequencing technology developed by Life Technologies and has been commercially available since 2006.
The ssUDNA is extracted from the bacteriophage that is released into the medium, and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heteroduplex DNA, that forms, consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP.
The Superscript II reverse transcriptase switches templates and continues transcribing to complete the 3’ end of the cDNA. This results in a full length cDNA containing the 5’ oligo-dT primer, cDNA transcribed from mRNA, and the 3’ universal priming site for second-strand synthesis.
Once hybridized, the universal primer can be extended, using the oligonucleotide probe as the template, to yield fully formed, target specific primers, which are then available to amplify the template in subsequent rounds of thermal cycling as per a standard PCR reaction.
Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red).
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