EzWay Direct ApoE Genotyping Kit

KOMABIOTECH INC.www.komabiotech.comEzWay Direct ApoE Genotyping Kit1. Catalog No. K05685002. No. of Applications 50 reactions3. Storage 1 year at -20°C, 1 month at 4°CAvoid repeated freeze and thaw.4. Contents Component Cat. No Volume Comment2X EzWay Direct TaqPCR MasterMixK0568010625ulMixture of Direct PCR Buffer,Taq DNA Polymerase, dNTP,MgCl 2 , Red dye and additive2.5X ApoE PrimerMix K0568501 500ul Primer mixture for ApoE geneDistilled Water K0568502 500ul PCR GradeSize marker K0570111S 50ul 100bp Ladder5. Description Human Apolipoprotein E (ApoE) is a 34 kDa glycoprotein that plays a central role inlipid metabolism and transportation. The ApoE gene is polymorphic with threecommon alleles, designated ε2, ε3, and ε4. These genes encode three ApoE proteinisoforms, E2 (Cys112/Cys158), E3 (Cys112/Arg158) and E4 (Arg112/Arg158), thatdiffer by cysteine-arginine interchanges at sites 112 and 158 in the polypeptide chain.The common ApoE isoforms exhibit variations in structure and function and areinvolved in several pathological processes. For instance, familial type IIIhyperlipoproteinemia is associated with the ApoE E2/E2 pheno/genotype, while theApoE E4/E4 pheno/genotype is associated with high cholesterol levels, coronaryartery disease, and Alzheimer’s disease.EzWay Direct ApoE Genotyping Kit contains Direct Taq PCR MasterMix, a newcocktail solution that facilitates polymerase chain reaction (PCR) without DNAisolation. Direct PCR Buffer in this MasterMix neutralizes inhibitors present in bloodand allows direct ApoE genotyping from blood without any DNA extraction. Also, theprimer mixture of ApoE genes enables to perform one-step Multiplex PCR.ApoE genotyping directly from bloodOne-step Multiplex PCR system with PrimerMixApoE primer mixture for E2 (Cys112/Cys158), E3 (Cys112/Arg158) and E4(Arg112/Arg158)Fast and simple method for genotyping without DNA purificationMinimize the risk of DNA loss or contaminationOptimized MasterMix type containing Taq enzyme, dNTP, Direct PCR Buffer,MgCl 2 , Red dye and additivesUseful detection of high cholesterol levels, cardiovascular disease orAlzheimer's diseaseFor research use only; not for use as a diagnostic1koma@komabiotech.comTel. 82-2-3662-7790 Fax. 82-2-3662-7730

KOMABIOTECH INC.www.komabiotech.com7. Result Table 1. Amplicons by APOE polymorphismsGenotype1 st , codon 112 a 2 nd , codon 158 a CommonAmpliconNt / AA b(bp)Nt / AA bAmplicon(bp)Amplicon(bp)ε2/ε2 TGC / Cys 113 TGC / Cys 253ε3/ε3 TGC / Cys 113 CGC / Arg 308ε4/ε4 CGC / Arg 444 CGC / Arg 308ε2/ε3 TGC / Cys 113TGC / CysCGC / Arg253308514ε2/ε4TGC / CysCGC / Arg113444TGC / CysCGC / Arg253308ε3/ε4TGC / CysCGC / Arg113444CGC / Arg 308a The mark of 1st and 2nd is order of mutation sequence in GenBank, and codonnumber is corresponded to each mutation.b Nt / AA is nucleotide / amino acid at mutation site.Direct ApoE genotyping from BloodBlood was amplified directly using EzWay Direct ApoE Genotyping Kit. One-stepMultiplex PCR enables to get the result just in 3 hours.For research use only; not for use as a diagnostic3koma@komabiotech.comTel. 82-2-3662-7790 Fax. 82-2-3662-7730

KOMABIOTECH INC.www.komabiotech.com8. Trouble Shooting Problem Probable Cause SolutionNo band Pipetting error Check pipettes.Problem with thermal cycleror PCR cycling conditionsCheck your thermal cycler and PCRcondition.Faint bandReagents and BloodPipetting errorProblem ReagentsCheck the storage conditions andthe expired date of kit reagents.Check your blood samples. If bloodsamples are obtained withanticoagulant treated container, youmust identify whether you havedrained blood the amountrecommended by the container’smanufacturer.Check the dispensed amount ofblood because of high viscosity ofblood.Check pipettes.Check the storage conditions andthe expired date of kit reagents.Smear bandCarry-over contaminationAgarose bufferPCR reagents or consumables maybe contaminated.We recommended to use TAEbuffer not TBE buffer, and highresolution agarose.4For research use only; not for use as a diagnostickoma@komabiotech.comTel. 82-2-3662-7790 Fax. 82-2-3662-7730