Peng H Z, Isaacson P G, Diss T C, Pan L X
Department of Histopathology, University College, London Medical School.
J Clin Pathol. 1994 Jul;47(7):605-8. doi: 10.1136/jcp.47.7.605.
To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples.
Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing.
Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA.
This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available.
建立一种简单可靠的聚合酶链反应（PCR）方法，用于从有限的组织病理学样本中对全基因组DNA进行随机扩增。
使用以随机六聚体为引物的两阶段PCR方案（RP-PCR），对从新鲜组织以及从存档石蜡组织切片中显微切割得到的单个淋巴滤泡中提取的微量基因组DNA进行扩增。将随机扩增的DNA样本用作特异性PCR扩增的模板。为检查RP-PCR的保真度，通过单链构象多态性（SSCP）或测序对特异性PCR扩增产物进行进一步分析。
使用一小部分RP-PCR模板库，成功进行了多项PCR分析，包括针对β珠蛋白基因、p53基因（外显子5-6、外显子7、外显子8-9和外显子7-9）以及重排的免疫球蛋白重链基因片段（VH框架3至JH和VH框架2至JH）的分析。与原始DNA产物相比，通过SSCP或测序在这些特异性PCR反应产物中未鉴定到人为突变。
该方法简单可靠，在仅有有限量组织可用时允许进行多项基因分析。
