Hot start PCR
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Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room (or colder) temperatures.[1][2] Many variations and modifications of the PCR procedure have been developed in order to achieve higher yields; hot start PCR is one of them.[3] Hot start PCR follows the same principles as the conventional PCR - in that it uses DNA polymerase to synthesise DNA from a single stranded template.[4] However, it utilizes additional heating and separation methods, such as inactivating or inhibiting the binding of Taq polymerase and late addition of Taq polymerase, to increase product yield as well as provide a higher specificity and sensitivity.[5] Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation.[6] Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures,[1][7] use of modified deoxyribonucleotide triphosphates (dNTPs),[8] and the physical addition of one of the essential reagents after denaturation.[9]
Through these additional methods, hot start PCR is able to decrease the amount of non-specific amplifications which naturally occur during lower temperatures – which remains a problem for conventional PCR. These modifications work overall to ensure that specific enzymes in solution will remain inactive or are inhibited until the optimal annealing temperature is reached.[10] Inhibiting formation of non-specific PCR products, especially in early cycles, results in a substantial increase in sensitivity of amplification by PCR. This is of utmost importance in diagnostic applications of PCR or RT-PCR.