A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome.
The rapid and reproducible identification of new cellular DNA sequences adjacent to known sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully identified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome.
Minami
M
M
INSERM U-370, CHU Necker, Paris, France.
Poussin
K
K
Bréchot
C
C
Paterlini
P
P
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D045647:DNA Glycosylases; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015894:Genome, Human; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D051981:Uracil-DNA Glycosidase; D016662:Virus Integration
D017931:DNA Primers; D004279:DNA, Viral; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase