Targeted gene walking by low stringency polymerase chain reaction: assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31.
Open Access
- 12 April 1994
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 91 (8) , 2975-2979
- https://doi.org/10.1073/pnas.91.8.2975
Abstract
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 degrees C) for the first few cycles followed by more cycles at high stringency (55 degrees C). This enables the amplification of a targeted DNA fragment along with other nontargeted fragments. 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). We 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, 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 III alpha (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 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 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.Keywords
This publication has 28 references indexed in Scilit:
- Temperature-sensitive mutations in the III–IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenitaCell, 1992
- Three brain sodium channel α-subunit genes are clustered on the proximal segment of mouse chromosome 2Genomics, 1991
- Assignment of a human skeletal muscle sodium channel α-subunit gene (SCN4A) to 17q23.1–25.3Genomics, 1991
- Hyperkalemic Periodic Paralysis and the Adult Muscle Sodium Channel α-Subunit GeneScience, 1990
- Localization of a human brain sodium channel gene (SCN2A) to chromosome 2Genomics, 1989
- Isolation of Single-Copy Human Genes from a Library of Yeast Artificial Chromosome ClonesScience, 1989
- A rat brain na+ channel α subunit with novel gating propertiesNeuron, 1988
- Primary structure of rat brain sodium channel III deduced from the cDNA sequenceFEBS Letters, 1988
- HLA-DQβ gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitusNature, 1987
- Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaScience, 1985