![]() The product sequences are then amplified in subsequent rounds of PCR (called the “tertiary reactions”) using another set of nested primers. This makes sequences bordered by a specific primer on one end and an AD primer on the other. The PCR program incorporates intermittent outer primer-favouring favouring cycles (called the “primary reactions”) between nested primer-favoured reactions (or “secondary reactions”). The two primer types have differing annealing temperatures. This technique comes in handy when we want to map where transfer DNA has inserted itself into the genome. They are added as higher concentrations than the outer primers so that after a few cycles they rule the roost and are producing more PCR product than the other primers. Next, an additional primer or pair of primers is added (our nested friends) which bind the sequence between the first set of primers, also called the “outer”, “arbitrary degenerate” (AD) or “border” primers. TAIL-PCR starts with thermal cycles of PCR and a pair of primers that are relatively far apart. This gives them far better discriminatory powers. Nested primers to the rescue! These primers bind within the sequence of the two “normal” primers and so “nest” cosily. In normal PCR, unless your primers are extremely specific, some non-specific binding and amplification of non-target sequences is very possible (even with hotstart PCR). These primers are not so-named because of a tendency to roost in intertwined branches, they’re just normal run-of-the-mill primers, but this does make for a rather curious mental image. TAIL-PCR makes use of “nested” or “specific” primers. This technique is great for finding out the regulatory sequences of a gene and to identify insertion sites in large genome tagging populations. You know you want to get to know someone so you ask a mutual friend to introduce you. Think of it as being rather like networking. Thermal asymmetric interlaced PCR or TAIL-PCR is used to sequence and analyse unknown DNA fragments that are adjacent to known sequences. What do bunnies, coins and PCR have in common? They all have tails! (ha ha!) ![]()
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