TARGETED GENE WALKING BY LOW STRINGENCY POLYMERASE CHAIN-REACTION - ASSIGNMENT OF A PUTATIVE HUMAN BRAIN SODIUM-CHANNEL GENE (SCN3A) TO CHROMOSOME-2Q24-31

We have developed a low stringency polymerase chain reaction (LSPCR) t o isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40-degrees-C) for the first f ew cycles followed by more cycles at high stringency (55-degrees-C). T his enables the amplification of a targeted DNA fragment along with ot her nontargeted fragments. High stringency (55-degrees-C) nested PCRs with end-labeled primers are then used to generate a ladder of radioac tive bands, which accurately identifies the targeted fragment(s). We p erformed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40-degrees-C followed by 27 cy cles at 55-degrees-C for primer annealing. Subsequently, using higher stringency (55-degrees-C) PCR with radiolabeled nested primers for 8 c ycles, we have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 27 0-bp region (exon) with homology to the rat brain sodium channel IIIal pha (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene codi ng the alpha-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, we have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase ch romosomes was used to sublocalize the SCN3A gene to chromosome at 2q24 -31. In conclusion, LSPCR is an efficient and sensitive method for tar geted gene walking and is also useful for the isolation of homologous genes in related species.