Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis.
Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.
Bjørheim
Jens
J
Department of Immunology, Section for Immunotherapy, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 0310, Norway.
Abrahamsen
Torveig Weum
TW
Kristensen
Annette Torgunrud
AT
Gaudernack
Gustav
G
Ekstrøm
Per O
PO
D000465:Algorithms; D000483:Alleles; D001331:Automation; D004247:DNA / Q000097:blood; D004252:DNA Mutational Analysis; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D017931:DNA Primers; D005819:Genetic Markers; D004247:DNA