Using universal degenerate primers for restriction digestion mapping by PCR.
In this study, a polymerase chain reaction (PCR) is developed to determine the restriction map without using restriction endonucleases. A 937 bp fragment of pUC 19 which contained one cut site for EcoRI and two recognition sites for PvuII was used as a model. The PCR was carried out using designed degenerate primers and the products were analyzed on 1.5% agarose gel. The number of cut sites, length of fragments and the arrangement of the fragments from 3' or 5' end of desired sequence were determined.
Sepehrizadeh
Zargham
Z
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. zsepehri@tums.ac.ir
Yazdi
Mojtaba Tabatabaei
MT
Shapourabadi
Mina Bahrololoumi
MB
D017931:DNA Primers; D015246:Deoxyribonuclease EcoRI / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping / Q000379:methods*
D017931:DNA Primers; D015246:Deoxyribonuclease EcoRI; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific