scholarly journals Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

1991 ◽  
Vol 88 (16) ◽  
pp. 7276-7280 ◽  
Author(s):  
P. M. Holland ◽  
R. D. Abramson ◽  
R. Watson ◽  
D. H. Gelfand
Vox Sanguinis ◽  
1997 ◽  
Vol 72 (3) ◽  
pp. 192-196 ◽  
Author(s):  
Dong-Feng Chen ◽  
Ladislav T. Pastucha ◽  
Hai-Yen Chen ◽  
Janos G. Kadar ◽  
Walter Stangel

2005 ◽  
Vol 83 (2) ◽  
pp. 147-165 ◽  
Author(s):  
François Vigneault ◽  
Régen Drouin

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo– DNA polymerase (Vent exo–) was developed. The optimal dosage of Vent exo– at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo– can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo– proves to be more efficient than Pyrococcus furiosus exo– (Pfu exo–) for this task. Vent exo– resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo–. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo– than for Pfu exo–, which is reflected by the sensibility of Vent exo– in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo– demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo– were established. Our results show that Vent exo– DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.Key words: Vent exo– DNA polymerase, Pfu exo– DNA polymerase, DNA sequence context, ligation-mediated polymerase chain reaction (PCR), PCR buffer.


2020 ◽  
Vol 48 (1) ◽  
pp. 62-72
Author(s):  
E. A. Ershova

Сalanoid copepods of the genus Pseudocalanus play an important role in the plankton communities of the Arctic and boreal seas, often dominating in numbers and constituting a significant proportion of the biomass of zooplankton. Despite their high presence and significance in the shelf plankton communities, species-specific studies of the biology of these are significantly hampered by extremely small morphological differences between them, especially at the juvenile stages, at which they are virtually indistinguishable. In this paper, we describe a new, routine and low-cost molecular method for identifying all Pseudocalanus species found in the Atlantic sector of the Arctic: the Arctic P. acuspes, P. minutus and the boreal P. moultoni and P. elongatus, and apply it to describe the relative distribution of these species in four locations of the Arctic and sub-Arctic. With this method, species-specific polymerase chain reaction (ssPCR), mass identification of individuals of any developmental stage, including nauplii, is possible. This method can serve as an excellent tool for studying the species-specific biology of this group, describing their life cycles, as well as monitoring changes in Arctic marine ecosystems under the influence of changing climate.


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