Targeted Gene Walking by Low Stringency Polymerase Chain Reaction: Assignment of a Putative Human Brain Sodium Channel Gene (SCN3A) to Chromosome 2q24-31

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-04, Vol.91 (8), p.2975-2979
• Main Authors: MALO, M. S, SRIVASTAVA, K, ANDRESEN, J. M, XIAO-NING CHEN, KORENBERG, J. R, INGRAM, V. M
• Format: Article
• Language: English
• Subjects: Animals; Annealing; Base Sequence; Biological and medical sciences; Brain; channels; chromosome 2; Chromosome Mapping; Chromosomes; Chromosomes, Human, Pair 2; Deoxyribonucleic acid; DNA; DNA Primers - chemistry; Exons; Fundamental and applied biological sciences. Psychology; Gels; gene mapping; Genes; Genes. Genome; Genetics; Humans; Hybridity; In Situ Hybridization, Fluorescence; man; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; nucleotide sequence; Nucleotides; Polymerase chain reaction; Polymerase Chain Reaction - methods; Rats; SCN3A gene; Sequence Alignment; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; sodium; Sodium channels; Sodium Channels - genetics; Walking
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.8.2975

We have developed a low stringency polymerase chain reaction (LSPCR) to 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⚬C) for the first few cycles followed by more cycles at high stringency (55⚬C). This enables the amplification of a targeted DNA fragment along with other nontargeted fragments. High stringency (55⚬C) nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). We performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40⚬C followed by 27 cycles at 55⚬C for primer annealing. Subsequently, using higher stringency (55⚬C) PCR with radiolabeled nested primers for 8 cycles, we have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel IIIα (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene coding the α-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 chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.