Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers.
A new procedure for sequence-independent PCR amplification of DNA fragments is described. DNA from pUC18 plasmid was used as a test DNA. It was digested with a frequently cutting restriction enzyme (Sau3A), generating sticky ends. The DNA was ligated to a synthetic, non-phosphorylated adaptor and subsequently amplified in a nested PCR using two oligonucleotides with sequences derived from the adaptor. As little as 1 fg of pUC18 DNA could be detected by this procedure. The product was analyzed on a gel and hybridized with a pUC18-specific probe. The sequence-independent nested PCR was repeated with different amounts of pUC18 DNA in the presence of an excess of non-specific DNA. In these experiments, pUC18 DNA fragments were amplified in a concentration-dependent manner. After hybridization with a digoxigenin dUTP-labelled pUC18 DNA probe, 1 fg of pUC18 DNA could still be detected. This method allows rapid screening of blood for low titred and mutated viruses in which primer binding sites are not conserved.
Schlayer
H J
HJ
Medizinische Universitätsklinik der Albert-Ludwigs Universität, Freiburg, Germany.
Peters
T
T
Preisler
S
S
Fehr
J
J
Gerok
W
W
Rasenack
J
J
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D014777:Virus Diseases / Q000175:diagnosis
D015345:Oligonucleotide Probes; D004247:DNA