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 T(m)'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 T(m) values. The ΔT applied is rather minute (1-1.5°C) and the temperature gradient quite shallow (e.g., 0.05°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°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.
| Original language | English |
|---|---|
| Pages (from-to) | 724-731 |
| Number of pages | 8 |
| Journal | Electrophoresis |
| Volume | 18 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 1997 |
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Keywords
- Capillary electrophoresis
- DNA electrophoresis
- N-Subtituted acrylamides
- Thalassemias
ASJC Scopus subject areas
- Clinical Biochemistry
Cite this
Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias. / Gelfi, Cecilia; Righetti, Pier Giorgio; Travi, Maurizio; Fattore, Silvia.
In: Electrophoresis, Vol. 18, No. 5, 05.1997, p. 724-731.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias
AU - Gelfi, Cecilia
AU - Righetti, Pier Giorgio
AU - Travi, Maurizio
AU - Fattore, Silvia
PY - 1997/5
Y1 - 1997/5
N2 - 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 T(m)'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 T(m) values. The ΔT applied is rather minute (1-1.5°C) and the temperature gradient quite shallow (e.g., 0.05°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°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.
AB - 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 T(m)'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 T(m) values. The ΔT applied is rather minute (1-1.5°C) and the temperature gradient quite shallow (e.g., 0.05°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°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.
KW - Capillary electrophoresis
KW - DNA electrophoresis
KW - N-Subtituted acrylamides
KW - Thalassemias
UR - http://www.scopus.com/inward/record.url?scp=0031001726&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031001726&partnerID=8YFLogxK
U2 - 10.1002/elps.1150180511
DO - 10.1002/elps.1150180511
M3 - Article
C2 - 9194597
AN - SCOPUS:0031001726
VL - 18
SP - 724
EP - 731
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 5
ER -