Surveyor™ Nuclease: A New Rapid and Effective Strategy for Detection of Single Nucleotide Changes in Alpha Globin Genes.

Alpha thalassemia is the most common globin gene disorder in the world and the disease is caused by mutations in either the alpha2 or alpha1 genes. Deletions of one (-α) or both (--) of these genes are the most common cause of alpha-thalassemia, while point mutations and small insertions/deletions are present but at a much lower frequency (5%). The number of point mutations described in these genes has been steadily increasing, with greater than 40 identified to date. Identifying these point mutations acquires importance as patients with non-deletional hemoglobin (Hb) H disease are more severely affected. Comprehensive evaluation of α globin genes is also essential while determining the molecular basis of the thalassemia intermedia phenotype. Mutations in the alpha globin gene can be detected either by direct DNA sequencing or by screening methods. Although small (about 1kb, 3 exons and 2 introns), the alpha-globin genes are duplicate and highly G-C rich, which makes them difficult to denature and therefore reducing sequencing efficiency and causing frequent artifacts. We have developed a simple and robust method based on mismatch-specific DNA endonuclease, SurveyorTM Nuclease. Genomic DNA control samples (n=21) harboring 12 different nucleotide changes characterized by direct DNA sequencing in either the alpha2 or alpha1 samples were used to validate this method. These samples were heterozygous or homozygous for each point mutation, or compound heterozygous for a deletion and the point mutation. Controls were amplified by gene specific primers designed by Primer3 software followed by digestion with SurveyorTM Nuclease. This endonuclease is a member of the CEL nuclease family of mismatch-specific nucleases derived from celery. It recognizes all insertions, deletions and base substitutions, and cleaves the mismatch of any heteroduplex DNA. After digestion, cleavage products are analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. All the 12 different nucleotide changes [α1 CD 14 TàC, α2 CD 104 TàG, α2 IVS II-55 TàG, α1 IVS II 141 TàC, α1 IVS I 117 GàT, α2 IVS I 1 GàA, Poly A (-AA), α1 CD 119 CàT, α2 IVS I 116, α2 CD 22 CàT, α2 CD 29 TàC, α2 +15 CàG] in the alpha globin genes were detected by this assay indicating a high sensitivity and specificity of this method. Mutation screening by SurveyorTM Nuclease is rapid (~ 5 hours), cost effective ($8/sample) and sensitive (100%). This assay in combination with the multiplex-PCR for screening the common alpha globin gene deletions will be the most comprehensive and economical approach for genetic diagnosis of alpha thalassaemia. Mismatch specific nucleases may have increasing application as a mutation detection strategy for human genetic diseases.