scholarly journals DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

2014 ◽  
Vol 5 ◽  
Author(s):  
Ashraf M. Elshawadfy ◽  
Brian J. Keith ◽  
H'Ng Ee Ooi ◽  
Thomas Kinsman ◽  
Pauline Heslop ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Chain Reaction ◽  
Thermococcus Kodakarensis ◽  
The Family ◽  
Polymerase Chain

Optimal conditions and specific characteristics of Vent exo– DNA polymerase in ligation-mediated polymerase chain reaction protocols

2005 ◽  
Vol 83 (2) ◽  
pp. 147-165 ◽  
Author(s):  
François Vigneault ◽  
Régen Drouin
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Pcr Amplification ◽  
Polymerase Activity ◽  
Optimal Dosage ◽  
Chain Reaction ◽  
Thermus Aquaticus ◽  
Thermococcus Litoralis ◽  
Polymerase Chain

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.

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

Gold nanoparticles alter Taq DNA polymerase activity during polymerase chain reaction

RSC Advances ◽  
2013 ◽  
Vol 3 (43) ◽  
pp. 20793 ◽  
Author(s):  
Samir Mandal ◽  
Maidul Hossain ◽  
T. Muruganandan ◽  
Gopinatha Suresh Kumar ◽  
Keya Chaudhuri
Keyword(s):  
Polymerase Chain Reaction ◽  
Gold Nanoparticles ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Parallel DNA Polymerase Chain Reaction (PD-PCR)

2016 ◽  
Author(s):  
vikash bhardwaj ◽  
Vikash bhardwaj ◽  
Kulbhushan Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Chain Reaction ◽  
Polymerase Chain
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