![]() This is one of the cardinal rules of PCR. The specificity of PCR is determined by the specificity of the PCR primers. Additional Tips to Increase Your specificity You can learn more about nested primers and their role in TAIL-PCR here and see a visual representation of the process here. Therefore, you can rest assured that your product is what you need it to be because of this extra level of selectivity. There may be several sequences within your starting material that contains a string of bases to which one set of primers will bind but it is statistically very unlikely that they will contain base strings capable of binding both set of primers. Having two pairs of primers acts like a double check. You then use a second set of primers, which have been designed as you would normally design primers, where they bind at or near the beginning of the target sequence. You run your PCR and end up with a product that contains both the target sequence and non-specific sequences. It uses two pairs of primers: the first set bind your target sequence but rather than binding closely to the beginning of the sequence, you design them to bind a little further away (by set we mean a forward and reverse primer). ![]() The end result is an impure product that may be unusable, depending on how much non-specific binding occurred and for what you need the PCR product. Unfortunately, this hope is more like a dream when your primers are less picky than you’d like when it comes to what sequences they’ll bind. The hope is that after 30-40 cycles, you’ll have an almost pure product with which to work. A pair of primers is used, called the forward and reverse primers, to ensure both strands of your sequence are amplified. In PCR, you design your primers to bind to the sequence you want amplified. Thankfully, a clever and surprisingly simple solution is at hand! A Quick Recap of the Basics Unless you’ve gotten your hands on some miraculously specific primers, amplification of only your target sequence without non-specific amplification can be very challenging. How to Obtain a Purer PCR Product and Reduce Non-specific Amplification ![]()
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