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PubMed1999

Dual tandem promoter elements containing CCAC-like motifs from the tetrodotoxin-resistant voltage-sensitive Na+ channel (rSkM2) gene can independently drive muscle-specific transcription in L6 cells.

cis-Elements in the -129/+124 promoter segment of the rat tetrodotoxin-resistant voltage-gated sodium channel (rSkM2) gene that are responsible for reporter gene expression in cultured muscle cells were identified by deletion and scanning mutations. Nested 5' deletion constructs, assayed in L6 myotubes and NIH3T3 cells, revealed that the minimum promoter allowing muscle-specific expression is contained within the -57 to +1 segment relative to the major transcription initiation site. In the context of the -129/+1 construct, however, scanning mutations in the -69/+1 segment failed to identify any critical promoter elements. In contrast, identical mutations in a minimal promoter (-57/+124) showed that all regions except -29/-20 are essential for expression, especially the -57/-40 segment, consistent with the 5' deletion analysis. Further experiments showed that the distal (-129/-58) and proximal promoter (-57/+1) elements can independently drive reporter expression in L6 myotubes, but not in NIH3T3 fibroblasts. This pair of elements is similar in sequence and contains Sp1 sites (CCGCCC), CCAC-like motifs, but no E-boxes or MEF-2 sites. The two segments form similarly migrating complexes with L6 myotube nuclear extracts in gel-shift assays. Critical elements within the distal promoter element were defined by 10 base pair scanning mutations in the -119 to -60 region in the context of the -129/+1 segment containing a mutated -59/-50 segment that inactivates the proximal promoter. Nucleotides in the -119/-90 region, especially -109/-100, were the most important regions for distal promoter function. We conclude that the -129/+1 segment contains two tandem promoter elements, each of which can independently drive muscle-specific transcription. Supershifts with antibodies to Sp1 and myocyte nuclear factor (MNF) implicate the involvement of Sp1, MNF, and other novel factors in the transcriptional regulation of rSkM2 gene expression.

Animals↗
PubMed2004

Genetic and biochemical analysis of cis regulatory elements within the keratinocyte enhancer region of the human papillomavirus type 31 upstream regulatory region during different stages of the viral life cycle.

Using linker scanning mutational analysis, we recently identified potential cis regulatory elements contained within the 5' upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the viral life cycle. For the present study, we extended the linker scanning mutational analysis to identify potential cis elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the viral life cycle. The linker scanning mutational analysis identified viral cis elements located in the KE region that regulate transcription in the presence and absence of any viral gene products or viral DNA replication and determine the role of host tissue differentiation on viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same cis regulatory elements at different stages of the HPV differentiation-dependent life cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify cis regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the viral life cycle. The variable recruitment of transcription factors to the same cis element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.

Base Sequence↗
PubMed1990

Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus.

Transcription from the promoter of mouse mammary tumor virus is subject to induction by several classes of steroid hormones as well as to repression by a negative regulatory element present in the long terminal repeats of proviral DNA. In order to characterize the functional elements of the promoter that in some way must respond to these regulatory signals, a number of promoter mutations were constructed, including a set of linker-scanning mutations across the entire promoter region. Analysis of these mutated promoters with a transient-transfection assay defined at least three mutation-sensitive promoter elements that are required for both basal and hormone-induced transcription. One mutation-sensitive region contains a TATA element located at approximately position -30 with respect to the start of transcription. A second mutation-sensitive region contains two 10-base-pair direct repeats located between positions -60 and -38, within which are embedded three copies of octamer-related sequences; complete disruption of this region of the promoter leads to a more severe decrease in transcription than do any of the linker-scanning mutations, suggesting that the repeated sequences may be at least partially functionally redundant. Gel electrophoresis mobility shift assays were used to demonstrate specific binding of a nuclear protein to this region of the promoter. A third mutation-sensitive region contains a binding site for nuclear factor 1 (NF-1) located between positions -77 and -63. Site-directed mutations in the NF-1-binding site which increase the apparent affinity of NF-1 for the promoter in vitro do not decrease the hormone dependence of transcription, suggesting that transcriptional activation mediated by steroid hormone-receptor complexes cannot be explained by facilitation or stabilization of the interaction of promoter sequences with NF-1 and consistent with the idea that binding of NF-1 is not rate determining in transcription from the mouse mammary tumor virus promoter. None of the promoter mutations functionally separates basal from glucocorticoid-induced transcription, suggesting that hormone induction does not make the promoter independent of any of the DNA-binding factors required for its basal activity.

Animals↗
PubMed1986

Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.

Base Sequence↗
PubMed2005

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods.

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.

DNA↗
PubMed1994

Exon scanning for mutation of the NF2 gene in schwannomas.

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.

Alternative Splicing↗
PubMed2005

Sodium channel mutations and susceptibility to heart failure and atrial fibrillation.

CONTEXT: Dilated cardiomyopathy (DCM), a genetically heterogeneous disorder, causes heart failure and rhythm disturbances. The majority of identified DCM genes encode structural proteins of the contractile apparatus and cytoskeleton. Recently, genetic defects in calcium and potassium regulation have been discovered in patients with DCM, implicating an alternative disease mechanism. The full spectrum of genetic defects in DCM, however, has not been established. OBJECTIVES: To identify a novel gene for DCM at a previously mapped locus, define the spectrum of mutations in this gene within a DCM cohort, and determine the frequency of DCM among relatives inheriting a mutation in this gene. DESIGN, SETTING, AND PARTICIPANTS: Refined mapping of a DCM locus on chromosome 3p in a multigenerational family and mutation scanning in 156 unrelated probands with DCM, prospectively identified at the Mayo Clinic between 1987 and 2004. Relatives underwent screening echocardiography and electrocardiography and DNA sample procurement. MAIN OUTCOME MEASURE: Correlation of identified mutations with cardiac phenotype. RESULTS: Refined locus mapping revealed SCN5A, encoding the cardiac sodium channel, as a candidate gene. Mutation scans identified a missense mutation (D1275N) that cosegregated with an age-dependent, variably expressed phenotype of DCM, atrial fibrillation, impaired automaticity, and conduction delay. In the DCM cohort, additional missense (T220I, R814W, D1595H) and truncation (2550-2551insTG) SCN5A mutations, segregating with cardiac disease or arising de novo, were discovered in unrelated probands. Among individuals with an SCN5A mutation 27% had early features of DCM (mean age at diagnosis, 20.3 years), 38% had DCM (mean age at diagnosis, 47.9 years), and 43% had atrial fibrillation (mean age at diagnosis, 27.8 years). CONCLUSIONS: Heritable SCN5A defects are associated with susceptibility to early-onset DCM and atrial fibrillation. Similar or even identical mutations may lead to heart failure, arrhythmia, or both.

Adult↗
PubMed2005

Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

Animals↗
PubMed2002

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system.

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).

Automation↗
PubMed1997

An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.

Bacteriophage lambda↗
PubMed2002

Reliability of DHPLC in mutational screening of beta-globin (HBB) alleles.

The inherited disorders of hemoglobin represent the most common Mendelian disease worldwide, with a higher prevalence among Mediterraneans, Asians, Africans, and Indians. Altered beta-globin sequences, causing either hemoglobinopathies or beta-thalassemia syndromes, are due to more than 200 different mutations in the beta-globin gene. Prevention programs based on postnatal and prenatal molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. We have developed a rapid and highly specific mutation screening test based on the denaturing high-performance liquid chromatography (DHPLC) system. The sensitivity and specificity of the method were tested on the full genomic region of the beta-globin gene in 30 normal Italian subjects and 40 heterozygous carriers in which 25 different beta-globin mutations had been previously characterized by multiplex-ARMS technique. The results showed DHPLC to be 100% sensitive and specific. All the 25 sequence alterations and two previously undetected polymorphisms were precisely identified with neither false positive nor false negative results. In addition, 12 compound heterozygous and four homozygous patients were successfully subjected to DHPLC. Overall, the method was able to rapidly identify the most common beta-globin mutations, accounting for more than 97% of beta-globin alleles in the Italian population. Compared to classical approaches of mutation screening, this method allows a rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.

Alleles↗
PubMed1996

Molecular scanning for mutations in the beta 3-adrenergic receptor gene in Nauruans with obesity and noninsulin-dependent diabetes mellitus.

We recently identified a mutation in the human beta 3-adrenergic receptor (beta 3AR) gene (codon 64 TGGTrp -> CGGArg; TRP64ARG) that associates with features of the insulin resistance syndrome and an earlier onset of noninsulin-dependent diabetes mellitus (NIDDM). We scanned the beta 3AR gene for mutations by single stranded conformational polymorphism analysis in 20 Nauruans with obesity and NIDDM. No mutations were identified. Sixty-five Nauruan subjects were genotyped for the TRP64ARG beta 3AR. All subjects were homozygous for the normal allele. We genotyped Samoans and Asians for the TRP64ARG beta 3AR. Seven of 52 Samoans were heterozygous for the mutant arginine allele (allele frequency, 0.07). Of these, 5 were diabetic and 2 were nondiabetic (by Fisher's exact test, P = 0.4). There were trends toward increased body mass indices, waist to hip ratios, and 2-h insulin levels during oral glucose tolerance tests in Samoans with the mutation; however, the limited number of subjects available for study precluded rigorous statistical analysis. The TRP64ARG beta 3AR was also detected in Chinese, Chinese Americans, and subjects from the Indian subcontinent. In conclusion, the TRP64ARG beta 3AR mutation or any other mutation in the beta 3AR gene is not a major contributor to genetic susceptibility to NIDDM and obesity likely in Nauruans.

Adult↗
PubMed1987

Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

Animals↗
PubMed2003

Scanning for mutations of the ryanodine receptor (RYR1) gene by denaturing HPLC: detection of three novel malignant hyperthermia alleles.

BACKGROUND: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. METHODS: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. RESULTS: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. CONCLUSION: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.

Alleles↗
PubMed2025

Robustness of Ancestral Sequence Reconstruction to Among-site and Among-lineage Evolutionary Heterogeneity.

Ancestral sequence reconstruction is typically performed using homogeneous evolutionary models, which assume that the same substitution propensities affect all sites and lineages. These assumptions are routinely violated: heterogeneous structural and functional constraints favor different amino acids at different sites, and these constraints often change among lineages as epistatic substitutions accrue at other sites. To evaluate how violations of the homogeneity assumption affect ancestral sequence reconstruction under realistic conditions, we developed site-specific substitution models and parameterized them using data from deep mutational scanning experiments on three protein families; we then used these models to perform ancestral sequence reconstruction on the empirical alignments and on alignments simulated under heterogeneous conditions derived from the experiments. Extensive among-site and -lineage heterogeneity is present in these datasets, but the sequences reconstructed from empirical alignments are almost identical when heterogeneous or homogeneous models are used for ancestral sequence reconstruction. Using models fit to deep mutational scanning data from distantly related proteins in which mutational effects are very different also has a minimal impact on ancestral sequence reconstruction. The rare differences occur primarily where phylogenetic signal is weak-at fast-evolving sites and nodes connected by long branches. When ancestral sequence reconstruction is performed on simulated data, errors in the reconstructed sequences become more likely as branch lengths increase, but incorporating heterogeneity into the model does not improve accuracy. These data establish that ancestral sequence reconstruction is robust to unincorporated realistic forms of evolutionary heterogeneity, because the primary determinant of ancestral sequence reconstruction is phylogenetic signal, not the substitution model. The best way to improve accuracy is therefore not to develop more elaborate models but to apply ancestral sequence reconstruction to densely sampled alignments that maximize phylogenetic signal at the nodes of interest.

Phylogeny↗
PubMed1997

Genetic testing for male infertility: a postulated role for mutations in sperm nuclear matrix attachment regions.

Numerous reports have suggested that disturbances in nuclear condensation may result in male infertility. This notion has been supported by the observation of infertile individuals with a decrease or absence of the male sperm-specific chromatin packaging protamine proteins. To date, no correlation between the absence of protamine proteins and a mutation within the coding regions of the protamine genes has been documented. To address this issue, PCR-based mutation scanning analysis has been performed across the human male haploid expressed PRM1-->PRM2-->TNP2 domain in several oligozoospermic infertile individuals. This analysis identified a candidate mutation in a region of contact with the sperm nuclear matrix from 2 of 5 affected individuals. This is the first report of a mutation scan covering the entire PRM1-->PRM2-->TNP2 locus in affected individuals.

Chromosomal Proteins, Non-Histone↗
PubMed2003

High-resolution genotyping by amplicon melting analysis using LCGreen.

BACKGROUND: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. METHODS: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. RESULTS: The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. CONCLUSIONS: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

Benzothiazoles↗
PubMed1994

Mutations in the phenylalanine hydroxylase gene: methods for their characterization.

Mutations in the phenylalanine hydroxylase (PAH) gene represent the root cause of PAH-deficient hyperphenylalaninemia. To date, more than 160 different mutations have been reported. Single-base substitutions and microdeletions account for the majority of molecular defects. This review provides a brief general introduction to various strategies for detection of PAH mutations, and summarizes our own methodological developments. We have established a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) for mutation scanning of the entire coding sequence and all exon/intron boundaries of the PAH. Systematic application of this method to the study of a large number of mutant chromosomes from hyperphenylalaninemic patients demonstrated a 98% diagnostic efficiency and a 100% mutation detection efficiency. We have created compromised PCR and DGGE conditions for simultaneous amplification and simultaneous mutation scanning of all PAH-coding fragments. This technique is convenient in a diagnostic setting and allows "same-day" DNA-based diagnosis of newborns with hyperphenylalaninemia. A further modification of the method allows unambiguous identification of known mutations, circumventing the cumbersome step of nucleotide sequencing.

DNA Mutational Analysis↗