Temperature‐gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction

Abstract

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.

Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis

“…The advantage of the psoralen cross-link over the GC clamp is most obvious if mutations in a GC-rich segment have to be tested. For the mutation in position 124, which is located in an 87% GC segment, it had been estimated earlier that a GC clamp had to be longer than 50 base pairs [19], whereas in the crosslinked fragment 7(+)Pt2(-) this and other mutations were easily detectable (CJ: Fig. 5C).…”

“…In order to separate crosslinked from noncross-linked DNA fragments and to analyze sequence variations within the crosslinked fragments in a single gel electrophoresis, tTGGE was used. This technique was applied in the parallel TGGE version [18] with many samples in narrow lanes similar to conventional gel electrophoresis. Figure 2A shows schematically that the first part of the electrophoretic analysis was carried out under denaturing conditions, i.e.…”

“…The mismatch decreases the thermal stability of the fragment and leads to a shift of the transition curve to a lower temperature. Most mutations were detected in TGGE if appropriate PCR fragments were chosen on the basis of thermodynamical calculations [19]. We used an algorithm for thermal denaturation of double-stranded nucleic acids that was based on the algorithm of Poland [20].…”

High-Throughput Mitochondrial Genome Screening Method for Nonmelanoma Skin Cancer Using Multiplexed Temperature Gradient Capillary Electrophoresis

“…Denaturing gradient gel electrophoresis (7 ), singlestrand conformation polymorphism analysis (8,9 ), conformation-sensitive gel electrophoresis (10,11 ), thermal gradient gel electrophoresis (12 ), enzymatic cleavage (13)(14)(15), and denaturing HPLC (DHPLC) (16 ) are examples of presequencing screening methods that have been developed to detect DNA variants. DHPLC (17,18 ), oligonucleotide sequencing arrays (19 ), and temporal temperature gradient gel electrophoresis (TTGE) have been used successfully as screening tools for mtDNA mutations (20 -24 ).…”

Analysis of thyroid stimulating hormone‐receptor mutations by temperature‐gradient gel electrophoresis

“…As a positive control-DNA for TGGE analysis a mutated fragment was constructed containing the mutation at codon 619 (Asp > Gly, gat > ggt). For mutagenesis, primers HST #1 and HST #2 were used in a recombinant PCR [6].TGGE was used as a screening method to search for point mutations [7]. To determine the optimal primer pair for TGGE analysis, melting profiles of the sequence of interest were calculated by the poland computer program (written by G. Steger, University of Diisseldorf, Germany).…”