Genotyping of human apolipoprotein E alleles by the new qualitative, microplate-based CASSI-detection assay.

Abstract:

:A new qualitative PCR product detection assay called competitive amplified single mutation detection by selective probe hybridization immunoassay (CASSI) was developed for genotyping the most common apolipoprotein E (apoE) polymorphisms. Single target DNA strands immobilized using biotin on streptavidin-coated microplates were hybridized in separate wells with two distinct, 5'-fluorescein isothiocyanate (FITC)-labeled oligonucleotides, complementary to either the 112Arg or 158Arg encoding site. With this assay, only correctly matched hybrids that form between probe and target DNA can be cleaved with the HhaI restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, the remaining microplate-adsorbed signal is measured colorimetrically by using anti-FITC, Fab-horseradish peroxidase conjugates. Our results show maximum intensity was detected when the respective probe hybridized incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cleavage); whereas, an intermediate signal was recorded at 50% complementarity (i.e., heterozygote alleles). With this assay, we could demonstrate a high prevalence of the apoE2 allele in patients suffering from coronary artery disease even though they displayed normal triglyceride and cholesterol levels. Corresponding results were obtained by CASSI compared with conventional restriction fragment-length polymorphism analysis.

journal_name

Biotechniques

journal_title

BioTechniques

authors

Köhler T,Rost AK,Purschwitz K,Vondran S,Remke H,Wagner O,Richter V

doi

10.2144/98251st03

subject

Has Abstract

pub_date

1998-07-01 00:00:00

pages

80-5

issue

1

eissn

0736-6205

issn

1940-9818

journal_volume

25

pub_type

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