title: Mutation detection of GJB2 using IsoCode and real-time quantitative polymerase chain reaction with SYBR green I dye for newborn hearing screening.
abstract: OBJECTIVE
  Recent developments in molecular genetics have opened a new era in genetic analysis accompanied by new concepts concerning genetic disorders. Although 30 genes responsible for nonsyndromic deafness have been discovered as of March 27, 2003, the connexin 26 gene (GJB2) is commonly found in cases of deafness of unknown origins. The GJB2 contains a predicted open reading frame of 785 base pairs, which makes it relatively easy to detect mutations. Accordingly, mutation analysis of GJB2 should be suitable for the screening of congenital deafness.
  
  
  METHODS
  Prospective study.
  
  
  METHODS
  IsoCode Stix is a useful device to isolate DNA from small samples of blood, which can be delivered from remote areas. To apply the detection of common mutations of GJB2 to hearing screening, DNA was extracted from several droplets of blood applied to the IsoCode device, and an allele-specific amplification method with real-time quantitative polymerase chain reaction was performed using GeneAmp 7700 with SYBR Green I dye.
  
  
  RESULTS
  DNA extracted from IsoCode was purified within 45 minutes, which was sufficient to detect the full sequence of GJB2. Four types of common GJB2 mutations were reliably detected within 2.5 hours.
  
  
  CONCLUSIONS
  IsoCode and real-time quantitative polymerase chain reaction will be promising tools for newborn screening of deafness genes in the future in DNA-based deafness screening, allowing early diagnosis of deafness and prompt training for language development.
authors.affiliation: Department of Medical Genetics, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kudo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Oshima
authors.forename: Shigeo
authors.initials: S
authors.lastname: Kure
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Matsubara
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Ikeda
mesh_terms: D052160:Benzothiazoles; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D004252:DNA Mutational Analysis; D003638:Deafness / Q000175:diagnosis* / Q000235:genetics*; D003959:Diamines; D005456:Fluorescent Dyes; D005820:Genetic Testing; D006320:Hearing Tests; D006801:Humans; D007231:Infant, Newborn; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D011804:Quinolines
chemical_list: D052160:Benzothiazoles; D017630:Connexins; D003959:Diamines; D005456:Fluorescent Dyes; C000607042:GJB2 protein, human; D009930:Organic Chemicals; D011804:Quinolines; D000072259:Connexin 26; C098022:SYBR Green I