Targeted gene walking by low stringency polymerase chain reaction: Assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31
- Massachusetts Institute of Technology, Cambridge, MA (United States)
- Univ. of California, Los Angeles, CA (United States)
The authors 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{degrees}C) for the first few cycles followed by more cycles at high stringency (55{degrees}C). Nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). They performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40{degrees}C followed by 27 cycles at 55{degrees}C for primer annealing. Subsequently, using higher stringency (55{degrees}C) PCR with radiolabeled nested primers for 8 cycles, they 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 III{alpha} (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, the authors putatively assign this sequence as a part of a gene coding 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, they 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.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 256467
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 8 Vol. 91; ISSN PNASA6; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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