Method for clone checking.
A new method for simple and fast clone checking is described. We combined the Pyrosequencing technology with a preprogrammed nucleotide dispensation strategy for fast analysis of DNA constructs. To test this method, the N-terminus region of plasmids constructed for the production of recombinant apyrase was analyzed. Of the ten plasmids tested, seven constructs were correct, two constructs showed one base deletion, and one construct showed deletion of a 195 bp fragment. The preprogrammed nucleotide dispensation strategy allowed the identification of the sequence downstream of the deletions. Thus, this method determines both the location and nature of possible artifacts.
Nourizad
Nader
N
Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm, Sweden.
Gharizadeh
Baback
B
Nyrén
Pål
P
D001081:Apyrase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D005822:Genetic Vectors / Q000032:analysis / Q000235:genetics; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion; D013997:Time Factors
D004274:DNA, Recombinant; D004247:DNA; D001081:Apyrase