Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kucukceylan N, Abdel-Nour M, Gewies A, Peters H, Kaufmann D, Buske A, Tinschert S, Nurnberg P:
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More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.
Am J Hum Genet 2000 Mar;66(3):790-818

The rapid detection of unknown mutations in nucleic acids.

Grompe M:
The task of identifying mutations in nucleic acid sequences is a vital component of research in mammalian genetics. With the advent of the polymerase chain reaction, several useful mutation detection techniques have evolved in recent years. The different methods have complementing strengths and a suitable procedure for virtually any experimental situation is now available.
Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland 97201.
Nat Genet 1993 Oct;5(2):111-7

Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.

Costes B, Girodon E, Ghanem N, Chassignol M, Thuong NT, Dupret D, Goossens M:
Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross- linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.
INSERM U91, Hopital Henri Mondor, Creteil, France.
Hum Mol Genet 1993 Apr;2(4):393-7

Mutation detection by denaturing gradient gel electrophoresis (DGGE).

Fodde R, Losekoot M:
The molecular analysis of genetic diseases relies on several technical approaches which allow genetic and physical mapping, characterization of the gene structure, expression studies, and identification of disease-causing mutations. Denaturing gradient gel electrophoresis (DGGE) allows the rapid screening for single base changes in enzymatically amplified DNA. The technique is based on the migration of double-stranded DNA molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent. In this review DGGE and the several modifications of the original protocol are presented. Moreover, its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single-strand conformation polymorphism. MGC-Department of Human Genetics, Sylvius Laboratorium, Leiden, The Netherlands.
Hum Mutat 1994;3(2):83-94I - 98357652

Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis.

Gille C, Gille A, Booms P, Robinson PN, Nurnberg P:
Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a "GC-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.
Institut fur Medizinische Genetik, Universitatsklinikum Charite, Berlin, Germany.
Electrophoresis 1998 Jun;19(8-9):1347-50UI - 88121658

Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis.

Lerman LS, Silverstein K:
Methods Enzymol 1987;155:482-501UI - 97220592

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene.

Macek M J, Mercier B, Mackova A, Miller PW, Hamosh A, Ferec C, Cutting GR:
More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations. Center for Medical Genetics, CMSC 1004, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914, USA.
Hum Mutat 1997;9(2):136-47

Nearly all single base substitutions in DNA fragments joined to a GC- clamp can be detected by denaturing gradient gel electrophoresis.

Myers RM, Fischer SG, Lerman LS, Maniatis T:
Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.
Nucleic Acids Res 1985 May 10;13(9):3131-45

A novel technique for rapid automated genotyping of DNA polymorphisms in the mouse.

Kuklin A, Davis AP, Hecker KH, Gjerde DT, Taylor PD:
The ability to rapidly and reliably genotype mice is an important concern. Traditional methods employ labour intensive and time consuming techniques such as test crossing, gel electrophoresis or nucleic acid hybridization. Here we show that a new molecular biology workstation, the WAVE DNA Fragment Analysis System, can easily resolve polymerase chain reaction (PCR) products that have small differences in their lengths. Analysis is fully automated and takes less than 7 min per sample. Approximately 200 samples can be analysed per day with only minutes of hands-on time after completion of the PCR. Genotyping with the WAVE DNA Fragment Analysis System is a fast and efficient method with minimal manual intervention. Copyright 1999 Academic Press. Transgenomic, Inc., 2032 Concourse Drive, San Jose, CA 95131, USA.
Mol Cell Probes 1999 Jun;13(3):239-42

Last modified: Tue Aug 31 18:46:25 CEST 2004

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