Exon concatenation to increase the efficiency of mutation screening by DGGE.
For genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE) requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were confirmed by using negative and positive controls for each of the DNAfragments analyzed.
Agirre
X
X
University of Navarra, Pamplona, Spain.
García-Delgado
M
M
Calasanz
M J
MJ
Larráyoz
M J
MJ
Novo
F J
FJ
Vizmanos
J L
JL
D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D005091:Exons / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics*
D004273:DNA, Neoplasm