Hot Start PCR - HotStart Polymerase Chain Reaction.
Hot Start PCR : An Introduction
Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation. By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield.
Hot Start PCR is commonly performed by using included chemical modifications, wax-barrier methods, and inhibition by a taq-directed antibody.
PCR or polymerase chain reaction uses DNA polymerases which were isolated from thermostable organisms such as the Taq enzyme, a eubacterial type I DNA polymerase, and Pfu, an archaeal type B DNA polymerase.
PCR amplifies DNA by multiple cycles of melting DNA at high temperature, annealing primers at a lower temperature, and then allowing polymerase extension at an intermediate temperature. Unfortunately, these thermophilic DNA polymerases show a very small but measurable polymerase activity at room temperature during assembly of the experiment.
This in-efficient DNA polymerase activity will catalyze the extension of any annealed 3' ends. After PCR amplification, the resulting product contains a mixture of specific and non-specific bands. Moreover, the 5'-3' exonuclease activity of the DNA polymerasel degrades any free 5' ends of partially annealed nucleic acids, destroying the primer and template substrates of the polymerase reaction. This results in a lower yield of the desired pcr product.
Hot Start PCR : Publications
Detection of bovine babesiosis in Mozambique by a novel seminested hot-start ... Related Articles
Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method.
Vet Parasitol. 2008 May 31;153(3-4):225-30
Authors: Martins TM, Pedro OC, Caldeira RA, do Rosário VE, Neves L, Domingos A
Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29-31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.
PMID: 18329810 [PubMed - in process]