PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).
Microdissection has proved to be a powerful tool in the construction of libraries from specific chromosome segments (11) which are poorly covered by existing RFLP markers. Microclones also represent starting points for finding genes of interest. However, their length (100 to 200 bp) can make their use as probes problematic and identifying them as coding sequence is difficult. We report here that microclones can be extended in vitro by a modified version of our original PCR walking method (10) which utilises oligo-cassettes and the solid phase biotin/streptavidin separation system. We have extended the microclone M54, derived by dissection from Xq27.2 to proximal Xq28 (12), in both directions for approximately 700 bp. Direct sequencing of these products revealed that M54 was located within an intron of the human gene encoding the neural cell adhesion molecule L1 (CAM-L1) which has been recently mapped to Xq28 (13). The extension of M54 also identified three exons of this gene. This information allowed subsequent amplification of a 2.4 kb cDNA molecule from fetal human brain mRNA which encodes most of human CAM-L1. Sequencing of this cDNA revealed a high degree of sequence conservation with the mouse homologue (14). This is the first description of extension of a human derived microclone by PCR mediated walking within total human genomic DNA. These results show that anonymous DNA sequences may be extended into coding or any sequence.
Rosenthal
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A
MRC Molecular Genetics Unit, Cambridge, UK.
MacKinnon
R N
RN
Jones
D S
DS
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics*; D016386:Chromosome Walking / Q000379:methods*; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D007438:Introns / Q000235:genetics; D039841:Leukocyte L1 Antigen Complex; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D019809:Streptavidin; D014960:X Chromosome
D001426:Bacterial Proteins; D015816:Cell Adhesion Molecules, Neuronal; D039841:Leukocyte L1 Antigen Complex; D009841:Oligonucleotides; D001710:Biotin; D019809:Streptavidin