A strategy for single site PCR amplification of dsDNA: priming digested cloned or genomic DNA from an anchor-modified restriction site and a short internal sequence.
Amplification of dsDNA by polymerase chain reaction (PCR) has been limited to those instances in which segments of known sequence flank the fragment to be amplified. A strategy for the PCR amplification of cloned or genomic dsDNA that necessitates sequence information from only a single short segment (single site PCR) has been devised. The region of known sequence may be located at any position within or adjacent to the segment to be amplified. The basic procedure for amplification consists of 1) digestion of dsDNA with one or more restriction enzymes, 2) ligation with a universal anchor adaptor and 3) PCR amplification using an anchor primer and the primer for the single site of known sequence. The anchor adaptor is designed in such a way as to facilitate the amplification of only those fragments containing the sequence of interest. We have demonstrated the utility of this technique by specifically amplifying and directly sequencing antibody variable region genes from cloned dsDNA and from genomic DNA.
Roux
K H
KH
Florida State University.
Dhanarajan
P
P
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011817:Rabbits
D004247:DNA; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI