[Detection of RB1 mutations by using quantitative fluorescent mutiplex PCR].
OBJECTIVE
To develop a half-automatic, simple, non-radioactive technique for rapid detection of mutation in the RB gene.


METHODS
Quantitative fluorescent multiplex PCR (QFM-PCR) involves amplification of the promoter region and all 27 exons of the RB1 gene with fluorescein labeled primers in multiplex sets. Primers were divided into multiplex sets of three to seven pairs of primers, also contained were internal control primers C4. Four external controls were also tested by using nullisomic, monosomic, diploid, and trisomic for RB1. The number of copies of a fragment in a test sample was calculated by comparing fluorescence intensity of the fragment with these standards. Fragment value detection and subsequent calculations were performed automatically by Fragment Manager 2.1 Software.


RESULTS
Small deletions, insertions and whole exon deletion in the RB1 gene were detected from genomic DNA. Sequencing analysis and RB tumor homozygous mutation comfirmed the defects detected by QFM-PCR.


CONCLUSIONS
The approach is a rapid, cost-effective initial method for screening patient samples. It has been used to identify approximately 50% positives of tested samples. The mutations shown were not only small deletion and insertion, but also the single copy exons heterozygous mutation in the RB1 gene which the previously used methods were unable to detect. These features make the technique an attractive approach to clinical diagnosis of gene defects.
Du
C
C
Genetics Service Center, Guangzhou General Hospital of PLA,Guangzhou 510080 P.R.China. wadewm@public.guangzhou.gd.cn
Gallie
B L
BL
D005453:Fluorescence; D016161:Genes, Retinoblastoma; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*