Microsatellite‐based cancer detection using capillary array electrophoresis and energy‐transfer fluorescent primers

Abstract

The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

“…The high throughput of these CAE microplates will facilitate rapid and inexpensive doublestranded restriction fragment screening for genetic diseases such as HHC. CAE microplates will also be valuable for single-stranded short tandem repeat analysis under denaturing conditions, following the methods recently developed by Wang and co-workers for forensic identification and cancer diagnosis (35,36).…”

Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

“…(LOH) Indicates LOH detected by method indicated with an allelic ratio variation of 20% which is considered diagnostically significant (Wang et al 1997 tion of 20% in allelic ratio is considered diagnostically significant with our methodology. The 20% value was determined through a detailed evaluation of the effect of PCR variability and fluctuations related to electrokinetic injection on the sensitivity and specificity of LOH analysis by CE (Wang et al 1997).…”

“…To translate this type of assay into a viable clinical tool, large-scale studies of the genetic changes associated with different tumors and different stages of tumor progression need to be undertaken with highthroughput modern instrumentation. As a first step, Wang et al (1997) validated microsatellite-based cancer detection using prototype capillary array electrophoresis apparatus and energy-transfer fluorescent primers. Di-, tri-, and tetra-nucleotide repeat markers, which have shown expansion, deletion, or LOH associated with primary bladder tumors, were examined on CAE using single-and two-color assays (Wang et al 1997).…”

“…Di-, tri-, and tetra-nucleotide repeat markers, which have shown expansion, deletion, or LOH associated with primary bladder tumors, were examined on CAE using single-and two-color assays (Wang et al 1997). The use of energy-transfer dye-labeled PCR primers allowed for the excitation of differentially labeled normal and tumor PCR amplicons at a common laser wavelength, while detecting non-overlapping emissions from the two samples (Wang et al 1997;Ju et al 1997).…”

Loss of heterozygosity and human telomerase reverse transcriptase (hTERT) expression in bronchial mucosa of heavy smokers

“…This limit is in general concordance with other previous studies. 27,28 To evaluate the allelic loss, normalized allelic ratios (R) were calculated as the equation in which R ¼ (A1/A2)/(N2/N1), where A1 and N1 are the peak heights of the smaller alleles (peak) and A2 and N2 of the larger alleles 29 in the bronchial sample (A) and in the peripheral blood (N).…”