Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

Abstract

We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (a subunit) gene sequence to chromosome 2q22-q23. When DNA from single, disected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-spedfic 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome mic ionolymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer sgiant advantages for other applications, such as cancer cytogenetics and linkage analysis.Assignment ofhuman genes or polymorphic DNA markers to specific chromosomal locations is a prerequisite for linkage analysis, location of individual genes, and the definition of their structure and function. In this communication we present a strategy for chromosome mapping that is direct, fast, and simple compared with traditional mapping techniques.Several methods, including somatic-cell genetics, in situ hybridization, and fluorescence-activated cell sorting of metaphase chromosomes, have been widely used to map structural genes and other identifiable DNA sequences onto chromosomes (1, 2). The most commonly used method for identifying the chromosomal locus of a specific human DNA sequence is in situ hybridization with radiolabeled probes. This approach is lengthy, labor-intensive, and often difficult to interpret because of cross-hybridization of the probe to related DNA sequences. Further, its mapping precision is limited by a variety of factors, including distortions in banding patterns caused by the preparation of specimens for hybridization and the spatial dispersion of isotopic signals captured on an emulsion overlay. Many of these drawbacks have been obviated by

Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain.

“…It is plausible that different human genes encoding sodium channel proteins are arranged within this region (32) since the probe derived from clone HB8 encodes a segment of amino acids that is 95% identical with rat brain subtypes I, II, and III. Indeed, brain-specific sodium channels were localized to a broad region of chromosome 2q21-33 by screening a panel of somatic cell hybrids followed by in situ hybridization (32), and the brain sodium channel II was mapped to chromosome 2q22-23 by using a chromosomal microdissection-PCR (33). Therefore, our fluorescent in situ hybridization measurements provide a more accurate localization.…”

Isolation of 2 novel RFLP markers and their localization at 2q35 by microdissection and subsequent enzymatic amplification

“…In one of the original CM-PCR methods, Han et al (1991) microdissected only one piece of a chromosomal segment and amplified the chromosomal DNA. To avoid possible contaminations or non-specific amplifications, they adopted the nested PCR.…”

“…For the purpose of direct mapping of the microclones which would represent RFLPs, we adopted the CM-PCR mapping method developed by Han et al (1991) and by Spielvoge! et al (1992).…”

“…For this purpose, chromosome fluorescence in situ hybridization (FISH) technique has generally been adopted , but FISH first needs the isolation of corresponding phage or cosmid clones with a minimal size of almost 1 kb, to be used as probes (Garson et al, 1987). An alternative mapping procedure, a chromosome microdissection and subsequent polymerase chain reaction (CM-PCR) method, has recently been developed by Han et al (1991) and by Spielvogel et al (1992), which involved PCR of the DNA from a defined microdissected chromosomal region using a set of clone-sequence-specific primers. This method provided rapid mapping of DNA fragments with a size as small as microclones.…”

Targeted gene walking by low stringency polymerase chain reaction: assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31.

“…Finally, three human brain sodium channel genes have now been subchromosomally localized (ref. 30; this report; unpublished observations mentioned above), providing candidate loci to be evaluated in families with genetic disorders.…”