Identification of apolipoprotein E polymorphisms using temperature cycled primer oligo base extension and mass spectrometry.
The isothermal Primer Oligo Base Extension (PROBE) reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnostic product detection as recently introduced by our group is modified to incorporate temperature cycling during the primer extension step, resulting in enhanced levels of diagnostic product generation. Utilizing temperature cycled PROBE, the identities of two apolipoprotein E polymorphisms (codons 112 and 158) for differentiation of epsilon 2/epsilon 3, epsilon 3/epsilon 3, epsilon 3/epsilon 4, and epsilon 4/epsilon 4 genotypes were simultaneously determined. Primers specific for each site are extended by a series of bases unique to the identity of that variable site, producing low mass diagnostic products (M(r) < 9000) highly amenable to detection by mass spectrometry. The temperature cycled PROBE method has yielded unambiguous and correct diagnoses for all samples tested thus far. The increased amount of diagnostic product generated per primer by the cycling method makes possible faster spectrum acquisition due to the increased signal intensity, critical for future automated measurement of such samples.
Little
D P
DP
Sequenom Instruments GmbH, Hamburg, Germany. danlittle@juno.com
Braun
A
A
Darnhofer-Demar
B
B
Köster
H
H
D001057:Apolipoproteins E / Q000097:blood / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature
D001057:Apolipoproteins E; D003062:Codon; D017931:DNA Primers