当前位置： SCI文献检索 > CLINICAL CHEMISTRY期刊下所有文献 > Nonfluorescent denaturing HPLC-based primer-extension method for allele-specific expression: application to analysis of mismatch repair genes.

Nonfluorescent denaturing HPLC-based primer-extension method for allele-specific expression: application to analysis of mismatch repair genes.

Abstract：

BACKGROUND:Altered germline expression of genes may represent a powerful marker of genetic or epigenetic predisposition to cancer or other diseases. METHODS:We developed and validated a method of nonfluorescent primer extension that uses a single dideoxynucleotide and denaturing HPLC (DHPLC) to analyze the relative allele expression. We devised 5 independent assays for measuring allele-specific expression (ASE) to exploit different markers of mismatch repair genes MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]. We initially confirmed method reproducibility with genomic DNA (gDNA) from individuals heterozygous for a frequent single-nucleotide polymorphism in the MLH1 gene. After this preliminary validation with gDNA, we confirmed assay reproducibility with cDNA templates from control individuals. Relative allele expression was estimated by comparing the heights of the peaks corresponding to the 2 alleles. Results obtained with gDNA templates were used to normalize cDNA results. RESULTS:With these DHPLC-based primer-extension assays, we detected and confirmed a 5-fold imbalance in MLH1 allele expression in a mutation-negative patient with hereditary nonpolyposis colorectal cancer and in another patient with a modest degree of imbalance in MLH1 expression. Among control individuals, the relative expression of MLH1 alleles displayed a narrow range of variation. CONCLUSIONS:Independent DHPLC-based primer-extension assays for measuring and confirming ASE can be developed for different sequence variants of interest. This DHPLC application provides a cost-effective method for detecting ASE in cases for which conventional screening fails to detect pathogenic mutations in candidate genes and may be applicable for confirming ASE revealed by other methods, such as those used for transcriptome-wide analyses. .

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Aceto GM,De Lellis L,Catalano T,Veschi S,Radice P,Di Iorio A,Mariani-Costantini R,Cama A,Curia MC

doi

10.1373/clinchem.2009.126300

subject

Has Abstract

pub_date

2009-09-01 00:00:00

pages

1711-8

issue

eissn

0009-9147

issn

1530-8561

pii

clinchem.2009.126300

journal_volume

pub_type

杂志文章

在线工具

Nonfluorescent denaturing HPLC-based primer-extension method for allele-specific expression: application to analysis of mismatch repair genes.