Polymerase chain reaction
2 Materials and methods
2.1 Cell isolation and DNA extraction
Granulocytes were isolated from peripheral blood of two unrelated individuals, A and B, on a Ficoll-Paque (G-S Chemical MFG. Corp.) gradient. High-molecular weight genomic DNA was extracted from the granulocytes by the salting out procedure [12]. Fragments of Bgl 11-digested genomic DNA were separated by electrophoresis in 1% agarose gel. A total of 34 gel slices, 2-5 mm in width, were cut out from the region, between 23 to 1.6 kb, which contained all theVH4 fragments. The DNA fragments then were purified with Geneclean beads (BIO 101 Inc., La Jolla, CA).
2.2 RFLP analysis
Southern blot analysis was performed by a standard procedure [13]. In brief, genomic DNA was digested with 5-10 units of Bgl IIIpg DNA overnight at 37 "C, followed by phenol-chloroform extraction and ethanol precipitation. Completely digested DNA was resuspended at a concentration of 1 pg/pl in distilled water, loaded at 10 pg per lane in 1% agarose gel, subjected to electrophoresis for 18 h at 40 volts, and transferred to a nylon membrane (Zeta-Probe membrane, Bio-Rad, Richmond, CA). The probe was a mixture of vH4 germ-line genes that was generated by PCR of DNA from individual A (see below). PCR products were initially purified from low-melting point agarose (SeaPlaque GTG agarose, FMC BioProducts, Rockland, ME) and extracted from the gel by Geneclean beads. Hybridization was carried out at 42ยฐC overnight with 32P-labeled PCR probes. The membrane was washed in 2 x SSC/O.l% SDS for 15 min at 22 "C, then in 0.5 x SSC/O.l% SDS for 30 min at 60 "C, and finally in 0.1 x SSC/O. 1% SDS for 1 h at 65 "C. The membrane was exposed to Hyperfilm-MP film (Amersham Int., Amersham, GB) at -70ยฐC.