title: High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay.
abstract: BACKGROUND
  Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.
  
  
  RESULTS
  A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.
  
  
  CONCLUSIONS
  Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.
authors.affiliation: Molecular Staging, Inc, 300 George Street, Suite 701, New Haven, CT 06511, USA. osamaa@molecularstaging.com
authors.forename: Osama A
authors.initials: OA
authors.lastname: Alsmadi
authors.forename: Carole J
authors.initials: CJ
authors.lastname: Bornarth
authors.forename: Wanmin
authors.initials: W
authors.lastname: Song
authors.forename: Michele
authors.initials: M
authors.lastname: Wisniewski
authors.forename: Jing
authors.initials: J
authors.lastname: Du
authors.forename: Joel P
authors.initials: JP
authors.lastname: Brockman
authors.forename: A Fawad
authors.initials: AF
authors.lastname: Faruqi
authors.forename: Seiyu
authors.initials: S
authors.lastname: Hosono
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Sun
authors.forename: Yuefen
authors.initials: Y
authors.lastname: Du
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Wu
authors.forename: Michael
authors.initials: M
authors.lastname: Egholm
authors.forename: Patricio
authors.initials: P
authors.lastname: Abarzúa
authors.forename: Roger S
authors.initials: RS
authors.lastname: Lasken
authors.forename: Mark D
authors.initials: MD
authors.lastname: Driscoll
mesh_terms: D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D004247:DNA / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D005165:Factor V / Q000235:genetics; D005260:Female; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D017354:Point Mutation / Q000235:genetics; D011516:Prothrombin / Q000235:genetics; D012189:Retrospective Studies
chemical_list: D015342:DNA Probes; D004270:DNA, Circular; D006680:HLA Antigens; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D004247:DNA