1 Answers
Hot start PCR is a modified form of conventional polymerase chain reaction that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room temperatures. Many variations and modifications of the PCR procedure have been developed in order to achieve higher yields; hot start PCR is one of them. 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. 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. Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation. Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures, use of modified deoxyribonucleotide triphosphates , and the physical addition of one of the essential reagents after denaturation.
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. 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.