We have recently developed a novel Immunoglobulin heavy chain gene rearrangement (IgH-R) assay that combines polymerase chain reaction (PCR) amplification and analysis in the same closed capillary tube using the Light...We have recently developed a novel Immunoglobulin heavy chain gene rearrangement (IgH-R) assay that combines polymerase chain reaction (PCR) amplification and analysis in the same closed capillary tube using the LightCycler System. IgHR can be identified by DNA melting curve analysis within 40 minutes after DNA preparation and amplification. To test the clinical utility of this new IgH-R assay for rapidly diagnosing cutaneous B-cell lymphomas, we prospectively analyzed 44 formalin-fixed, paraffin-embedded tissues suspected of Bcell malignant lymphoma: skin (n = 31), lymph node (n = 7), stomach (n = 3), spleen (n = 1), colon (n = 1), and soft tissue (n = 1). We detected IgH-R in 12 DNA samples, including 8 skin biopsies, with the following diagnoses: B-cell chronic lymphocytic leukemia (n = 4), extranodal marginal zone Bcell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 2), Burkitt lymphoma (n = 1), and precursor B-lymphoblastic lymphoma(n = 1). DNAmelting curve analysis, comparedwith polyacrylamide gel electrophoresis, achieved a sensitivity equal to 92.3%and a specificity equal to 100%. There was a single false negative result because DNA melting curve analysis could not detect less than 10.0%clonal B-cells. We conclude that this new, rapid PCR assay for detecting IgH-R based on DNA melting curve analysis can be clinically useful for confirming the initial diagnosis of B-cell malignant lymphoma.展开更多
为研究古菌Pol D DNA聚合酶两个亚基的功能及其相互作用,对重组超嗜热硫还原古菌Thermococcus sp.4557 Pol D DNA聚合酶小亚基DP1和大亚基DP2进行柱层析纯化,采用荧光标记核酸结合聚丙烯酰胺凝胶电泳实验对两个亚基体外功能进行研究。...为研究古菌Pol D DNA聚合酶两个亚基的功能及其相互作用,对重组超嗜热硫还原古菌Thermococcus sp.4557 Pol D DNA聚合酶小亚基DP1和大亚基DP2进行柱层析纯化,采用荧光标记核酸结合聚丙烯酰胺凝胶电泳实验对两个亚基体外功能进行研究。结果表明,DP2具有DNA复制延伸活性,DP1具有3′→5′外切核酸酶活性;DP1在与DP2的浓度比高于0.3∶1时可降解DP2的延伸产物;DP2对DP1的外切核酸酶活性有促进作用,Sld5和Psf1蛋白复合体(Sld5 and Psf1 Complex, GINS 51)对DP1的外切核酸酶活性有抑制作用。古菌Pol D DNA聚合酶大小亚基的功能及其相互作用对DNA复制机制的进一步认识有借鉴意义。展开更多
文摘We have recently developed a novel Immunoglobulin heavy chain gene rearrangement (IgH-R) assay that combines polymerase chain reaction (PCR) amplification and analysis in the same closed capillary tube using the LightCycler System. IgHR can be identified by DNA melting curve analysis within 40 minutes after DNA preparation and amplification. To test the clinical utility of this new IgH-R assay for rapidly diagnosing cutaneous B-cell lymphomas, we prospectively analyzed 44 formalin-fixed, paraffin-embedded tissues suspected of Bcell malignant lymphoma: skin (n = 31), lymph node (n = 7), stomach (n = 3), spleen (n = 1), colon (n = 1), and soft tissue (n = 1). We detected IgH-R in 12 DNA samples, including 8 skin biopsies, with the following diagnoses: B-cell chronic lymphocytic leukemia (n = 4), extranodal marginal zone Bcell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 2), Burkitt lymphoma (n = 1), and precursor B-lymphoblastic lymphoma(n = 1). DNAmelting curve analysis, comparedwith polyacrylamide gel electrophoresis, achieved a sensitivity equal to 92.3%and a specificity equal to 100%. There was a single false negative result because DNA melting curve analysis could not detect less than 10.0%clonal B-cells. We conclude that this new, rapid PCR assay for detecting IgH-R based on DNA melting curve analysis can be clinically useful for confirming the initial diagnosis of B-cell malignant lymphoma.
文摘为研究古菌Pol D DNA聚合酶两个亚基的功能及其相互作用,对重组超嗜热硫还原古菌Thermococcus sp.4557 Pol D DNA聚合酶小亚基DP1和大亚基DP2进行柱层析纯化,采用荧光标记核酸结合聚丙烯酰胺凝胶电泳实验对两个亚基体外功能进行研究。结果表明,DP2具有DNA复制延伸活性,DP1具有3′→5′外切核酸酶活性;DP1在与DP2的浓度比高于0.3∶1时可降解DP2的延伸产物;DP2对DP1的外切核酸酶活性有促进作用,Sld5和Psf1蛋白复合体(Sld5 and Psf1 Complex, GINS 51)对DP1的外切核酸酶活性有抑制作用。古菌Pol D DNA聚合酶大小亚基的功能及其相互作用对DNA复制机制的进一步认识有借鉴意义。
