High‐resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy‐transfer fluorescent primers

Abstract

Short tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5' end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). M13 A-track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high-resolution, high-speed and high-throughput STR typing of single-stranded DNA fragments using capillary array electrophoresis.

High-Precision Genotyping by Denaturing Capillary Electrophoresis

“…for alleles 7-9 of 0.18 nucleotide, but a S.D. of 0.34 nucleotide for allele 6 (Wang et al 1996). By use of a similar polymer mix at room temperature, Mansfield et al (1996) report S.D.s for the THO1 allelic ladder fragments ranging from 0.10 to 0.14 nucleotide.…”

“…To increase the accuracy in sizing, it might be possible to calculate a normalizing factor (Mansfield et al 1996) or, alternatively, energy-transfer fluorescent dye-labeled DNA primers Wang et al 1995) that exhibit only very minor size differences could be used. More important than the absolute sizing accuracy, however, are highly reproducible analyses (Pritchard et al 1995;Wang et al 1996). These analyses should provide consistent genotyping for the same allelic STR, when using similar instrumentation and run conditions, independent from the genomic source and from run-to-run.…”

“…We selected two different STR systems to evaluate the performance of polymers for separation and precision: (1) an allelic ladder derived from the human THO1 locus, which is often used to describe the characteristics of a particular separation system (Williams et al 1994;Butler et al 1995;Wang et al 1995;Mansfield et al 1996;Wang et al 1996), and (2) DNAs from four different CEPH families, amplified with Genethon dinucleotide repeat markers (Dib et al 1996).…”

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).…”

DNA typing in thirty seconds with a microfabricated device

“…To improve throughput, arrays of capillaries have been operated in parallel (14)(15)(16)). An analysis of the quadruplex STR system CTTv was reported on a 5-column array device with a throughput of 5 samples in 50 min (17). Also, a 48-column array system has been described where 48 samples could be analyzed in parallel in 1 h (18).…”