Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.
Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.
Department of Cell & Molecular Biology, Faculty of Biotechnology & Biomolecular Sciences, Putra-University Malaysia, 43400 UPM-Serdang, Selangor Darul Ehsan, Malaysia.
Tran
T
Thanh
Vu Thi Quynh
VT
Chi
Mohd Puad
MP
Abdullah
Hishamuddin
H
Omar
Suhaimi
S
Napis
D008026:Ligation / Q000379:methods*; D058086:Microalgae / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification*
D012273:Ribulose-Bisphosphate Carboxylase