Mass allele detection (MAD) of rare 5-HT1A structural variants with allele-specific amplification and electrochemiluminescent detection.
A strategy is described that exploits allele-specific amplification (ASA-PCR) and electrochemiluminescence (ECL) detection technology to rapidly and cheaply screen large numbers of DNAs arranged in pooled matrices in order to identify individual nucleotide sequence variants. To demonstrate this strategy, a large genomic DNA collection was screened for two nucleotide variants in the 5-HT1A serotonin receptor gene and individual heterozygotes were identified. Conversion of two SSCP variants to allele-specific PCR polymorphisms was accomplished, and PCR product capture and ECL detection were enabled by the covalent addition of biotin to allele-specific PCR primers and ruthenium to the nonspecific PCR primer. A two-level DNA pooling strategy was used to reduce the number of individual PCR reactions required. Pooling experiments established that ASA-PCR with ECL detection is sufficiently sensitive to reproducibly detect a single specific allele in the presence of a 40-fold excess of genomic DNA from individuals negative for the specific allele. The detection sensitivity of the ECL device and the design of the pooled DNA arrays reduced the number of PCRs required to detect the rare individuals with the variant sequences by approximately 90%. This strategy is called mass allele detection (MAD).
Bergen
A
A
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland, USA.
Wang
C Y
CY
Nakhai
B
B
Goldman
D
D
D000483:Alleles; D001483:Base Sequence; D001710:Biotin; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005006:Ethnicity / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D006579:Heterozygote; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics*; D044263:Receptors, Serotonin, 5-HT1; D012680:Sensitivity and Specificity
D017931:DNA Primers; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D001710:Biotin