Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers
1992
Abstract 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 ( Sau 3A), 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 digoxigenine 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.
Keywords:
- Virology
- Molecular biology
- Multiple displacement amplification
- Ligase chain reaction
- Polymerase cycling assembly
- Biology
- Multiplex ligation-dependent probe amplification
- Sequencing by ligation
- Genetics
- Primer (molecular biology)
- In vitro recombination
- Primer dimer
- DNA extraction
- Genomic library
- Sticky and blunt ends
- Correction
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