Direct sequencing of affinity-captured amplified human DNA application to the detection of apolipoprotein E polymorphism.
We describe a method for the direct sequencing of DNA amplified by the polymerase chain reaction (PCR). Biotin is introduced into one strand of the amplified DNA using a 5'-biotinylated PCR primer. The synthesized fragment is captured on an avidin-matrix and rendered single stranded, whereafter the nucleotide sequence of the immobilized strand is determined by the chain termination method. The method involves few and simple operations and is thus applicable to the analysis of human genes for routine diagnostic purposes. Here we applied the method for determination of the three-allelic polymorphism of the apolipoprotein E (apo E) gene. We were able to correctly identify the alleles in both homozygous and heterozygous samples.
Syvänen
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Orion Corporation, Genetic Engineering Laboratory, Helsinki, Finland.
Aalto-Setälä
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Kontula
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Söderlund
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H
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D001057:Apolipoproteins E / Q000235:genetics*; D001345:Autoradiography; D001483:Base Sequence; D001710:Biotin; D004277:DNA, Single-Stranded / Q000032:analysis; D030342:Genetic Diseases, Inborn / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D000345:Affinity Labels; D000596:Amino Acids; D001057:Apolipoproteins E; D004277:DNA, Single-Stranded; D001710:Biotin