A novel method to isolate the common fraction of two DNA samples: hybrid specific amplification (HSA).
Hybrid specific amplification (HSA) is a novel simple method elaborated in order to isolate the common fraction of two DNA samples while avoiding the background due to repeated sequences. The method is based on the suppressive PCR principle, associated with a Cot1 pre-hybridization step. In recent work we demonstrated that hyperprolificity observed in Booroola ewes is associated with a mutation in the bone morphogenetic protein receptor IB gene (BMPR-IB). We applied HSA between ovarian cDNA and DNA from four BAC clones containing BMPR-IB in order to test for the presence of other genes expressed in ovary and to isolate additional BMPR-IB exon sequences. Of the 460 clones obtained, none contained repeated sequences. We successfully obtained 37 clones representing the major part of BMPR-IB coding sequence, together with 5'- and 3'-UTR sequences. Here we have successfully applied HSA to a particular tissue, but it should be possible to trap the common fraction of two DNA samples, whatever their nature.
Lecerf
F
F
Laboratoire de Génétique Cellulaire, INRA Toulouse, Chemin de Borde Rouge, BP 27, F-31326 Castanet Tolosan, France. flecerf@toulouse.inra.fr
Foggia
L
L
Mulsant
P
P
Bonnet
A
A
Hatey
F
F
D000818:Animals; D052005:Bone Morphogenetic Protein Receptors, Type I; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D012756:Sheep
D018076:DNA, Complementary; D017978:Receptors, Growth Factor; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D052005:Bone Morphogenetic Protein Receptors, Type I