CAJ | 학술논문

目的建立非变性毛细管电泳分离乙肝病毒特异性巢式PCR产物方法,用于乙肝病毒B基因型和C基因型判断.方法建立线性聚丙烯-聚乙二胺-TBE非变性毛细管电泳方法,分离母婴阻断失败患儿血清中乙肝病毒基因型特异性巢式PCR的扩增产物,采用表观分子量鉴定B、C基因型特异性和非特异性扩增产物.结果毛细管电泳条件为30.2cm×50μm毛细管,分离缓冲液为2％(w/v)线性聚丙烯酰胺+0.4％(w/v)聚乙二醇+lxTBE缓冲液(pH8.3).在9kV电压下12分钟内可分离DNA分子量梯度标准品(50 bp至300 bp)及PCR扩增产物,表观分子量与电泳迁移时间的曲线适合度大于0.9999.B型扩增产物表观分子量范围为282bp至285bp,非特异性扩增产物为276bp至280bp.C型为ll9bp至120bp,当前扩增条件下不产生非特异性扩增产物.结论非变性毛细管电泳能够方便和准确地判断非特异性的B型扩增产物.
목적 건립비변성모세관전영분리을간병독특이성소식PCR산물방법,용우을간병독B기인형화C기인형판단.방법 건립선성취병희-취을이알-TBE비변성모세관전영방법,분리모영조단실패환인혈청중을간병독기인형특이성소식PCR적확증산물,채용표관분자량감정B、C기인형특이성화비특이성확증산물.결과 모세관전영조건위30.2cm×50μm모세관,분리완충액위2％(w/v)선성취병희선알+0.4％(w/v)취을이순+lxTBE완충액(pH8.3).재9kV전압하12분종내가분리DNA분자량제도표준품(50 bp지300 bp)급PCR확증산물,표관분자량여전영천이시간적곡선괄합도대우0.9999.B형확증산물표관분자량범위위282bp지285bp,비특이성확증산물위276bp지280bp.C형위ll9bp지120bp,당전확증조건하불산생비특이성확증산물.결론 비변성모세관전영능구방편화준학지판단비특이성적B형확증산물.
Objective To establish the non-denaturant capillary electrophoresis method to separate nested PCR products with HBV genotype specific primers,and confirm HBV genotypes B and C.Methods The non-denaturant capillary electrophoresis method with linear polyacrylamide-polyethylene-TBE buffer system was first established.The nested PCR method with HBV genotype specific primers was used to amply HBV DNA segments from serums of children who are failed in prevention of mother-to-children transmission.The apparent molecular weights indicate.specific and non-specific amplicons of genotype B and C.Results Capillary electrophoresis conditions applied a 30.2cm x 50μm i.d.fused silica capillary,running buffer containining 2％(w/v) linear polyacrylamide,0.4％ (w/v) polyethylene and 1 × TBE buffer (pH8.3).Separation of 50-bp DNA step ladder and PCR products were completed within 12 min under 9 kV.The goodness-of-fit between molecular weights and migration times was over 0.9999.Apparent molecular weights of specific amplification products for genotype B range from 282 bp to 285 bp,non-specific products range 276 bp to 280 bp.For genotype C,the products were 119 bp to 120 bp,and no non-specific amplification was observed under current conditions.Conclusion The non-denaturant capillary electrophoresis method could conveniently separate and identify non-specific amplification products of genotype B.