Screening for mutations in kidney-related genes using SURVEYOR nuclease for cleavage at heteroduplex mismatches.
SURVEYOR is a new mismatch-specific plant DNA endonuclease that is very efficient for mutation scanning in heteroduplex DNA. It is much faster, cheaper, more sensitive, and easier to perform than other "traditional" mutation detection methods such as single-strand conformation polymorphism analysis, denaturing high-performance liquid chromatography, heteroduplex analysis, and phage resolvases. This is the first comprehensive report on the use of SURVEYOR for screening genes implicated in a spectrum of inherited renal diseases. Of the 48.2 kb screened, 44 variations were identified, accounting for one variation per 1.1 kb. The re-sequencing of multiple samples did not reveal any variation that had not been identified by SURVEYOR, attesting to its high fidelity. Additionally, we tested this enzyme against 15 known variants, 14 of which it identified, thus showing a sensitivity of 93%. We showed that the genetic heterogeneity of renal diseases can be easily overcome using this enzyme with a high degree of confidence and no bias for any specific variations. We also showed for the first time that SURVEYOR does not demonstrate any preference regarding mismatch cleavage at specific positions. Disadvantages of using SURVEYOR include enhanced exonucleolytic activity for some polymerase chain reaction products and less than 100% sensitivity. We report that SURVEYOR can be used as a mutation detection method with a high degree of confidence, offering an excellent alternative for low-budget laboratories and for the rapid manipulation of multiple genes.
Voskarides
Konstantinos
K
Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.
Deltas
Constantinos
C
D020137:Base Pair Mismatch; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004720:Endonucleases / Q000378:metabolism*; D005820:Genetic Testing / Q000379:methods; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000502:physiology*; D007674:Kidney Diseases / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D009692:Nucleic Acid Heteroduplexes; D010940:Plant Proteins; D004720:Endonucleases