Temperature‐programmed capillary electrophoresis for the analysis of high‐melting point mutants in thalassemias

Abstract

The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The deltaT applied is rather minute (1-1.5 degrees C) and the temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60-62 degrees C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N-acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.

Evaluation of denaturing conditions in analysis of DNA variants applied to multi‐capillary electrophoresis instruments

“…The use of denaturant gradients with Joule heating and temperature zone have been applied to single capillary instruments [18 -20]. However, these strategies did not improve the sensitivity and specificity of the mutation analysis as compared to CDCE [1,21]. In the present setup, the use of gradients and cycling in the multi-capillary system was adapted for an entirely different reason: The original purpose of the gradient was to eliminate local temperature differences in the capillary chamber.…”

DNA Diagnostics by Capillary Electrophoresis

“…The timeprogrammed temperature approach has been applied for detection of point mutations in the human genome. 613,648,649 Gelfi et al used DGCE with temperature programming for detection of mutations in cystic fibrosis transmembrane conductance regulator gene 613,648 and β-thalassemia mutations in β-globin gene. 649 Several papers focused on development of high-throughput automated instruments with precise control of spatial and temporal temperature gradients.…”

Molecular analysis of unknown β-globin gene mutations using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique and its application in Thai families with β-thalassemias and β-globin variants

“…It should be more economical to initially screen the region where mutations are located by a simple method before performing a sitespecific sequencing analysis. Previously, several techniques for mutation screening of the h-globin gene have been developed including denaturing gradient gel electrophoresis (DGGE) [21], chemical cleavage of mismatched (CCM) [22], dideoxy fingerprinting (ddF) [23] and temperature gradient gel electrophoresis (TGGE) [24]. However, some practical disadvantages were encountered.…”

“…Therefore, there is a need for an inexpensive, rapid, molecular detection method before sequencing analysis. Several screening techniques for unknown mutations exist, and they include denaturing gradient gel electrophoresis (DGGE) [21], chemical cleavage of mismatched DNA (CCM) [22], dideoxy fingerprinting (ddF) [23], temperature gradient gel electrophoresis (TGGE) [24] and single strand conformation polymorphism (SSCP) [25]. The application of the PCR-SSCP technique for analysis of the h-globin gene mutations has not been thoroughly explored.…”