High-speed, multicolor fluorescent two-dimensional gene scanning.

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.

BRCA1 gene sequence variation in centenarians.

With the ample gene sequence information that has become available with the human genome project virtually completed, it has become possible to identify functional gene variants and their frequencies in elderly populations with different aging-related characteristics. Such a genetic epidemiological approach could lead to new insights with respect to the basic mechanisms of aging and longevity as well as the identification of new targets to prevent or retard some of the late-age adverse effects. Using our recently developed two-dimensional gene scanning (TDGS) technology platform we demonstrate the feasibility of this approach by screening two different populations of centenarians for polymorphic variation in the BRCA1 breast cancer susceptibility gene, one of the many genes involved in genome maintenance. The initial results obtained with this approach suggest differences in BRCA1 genotype frequencies between the centenarian populations and controls.

Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.

Somatic mutations in mtDNA have recently been identified in colorectal tumours. Studies of oncocytic tumours have led to hypotheses which propose that defects in oxidative phosphorylation may result in a compensatory increase in mitochondrial replication and/or gene expression. Mutational analysis of mtDNA in thyroid neoplasia, which is characterised by increased numbers of mitochondria and is also one of the most common sites of oncocytic tumours. has been limited to date. Using the recently developed technique of two-dimensional gene scanning, we have successfully examined 21 cases of thyroid tumours, six cases of non-neoplastic thyroid pathology, 30 population controls, nine foetal thyroid tissues and nine foetal tissues of non-thyroid origin, either kidney or liver. We have identified three different somatic mutations (23%) in papillary thyroid carcinomas. In addition, we have found significant differential distributions of mtDNA sequence variants between thyroid carcinomas and controls. Interestingly, these variants appear to be more frequent in the genes which encode complex I of the mitochondrial electron transport chain compared to normal population controls. These findings suggest first, that somatic mtDNA mutations may be involved in thyroid tumorigenesis and second, that the accumulation of certain non-somatic variants may be related to tumour progression in the thyroid.

A highly accurate, low cost test for BRCA1 mutations.

The hereditary breast and ovarian cancer syndrome is associated with a high frequency of BRCA1 mutations. However, the widespread use of BRCA1 testing has been limited to date by three principal concerns: the fear of loss of health and life insurance, the uncertain clinical value of a positive test result, and the current lack of an inexpensive and sensitive screening test for BRCA1 mutations. We have developed an inexpensive system for gene mutational scanning, based on a combination of extensive multiplex PCR amplification and two dimensional electrophoresis. The efficiency of this system, as a screening test for BRCA1 mutations, was evaluated in a panel of 60 samples from high risk women, 14 of which contained a previously identified mutation in BRCA1. All 14 mutations were identified, as well as an additional five that had previously escaped detection. In addition to the 19 mutations, a total of 15 different polymorphic variants were scored, most of which were recurring. All were confirmed by nucleotide sequencing. The cost of screening per sample was calculated to be approximately US$70 for the manual technique used in this study, and may be reduced to approximately US$10 with the introduction of commercially available PCR robotics and fluorescent imaging. Implementation of this method of mutation screening in the research and clinical setting should permit rapid accrual of quantitative data on genotype-phenotype associations for the evaluation of diagnostic testing.

Background mutations and polymorphisms in lacZ-plasmid transgenic mice.

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.

Two-dimensional gene scanning: exploring human genetic variability.

Current methods for mutation detection are not optimized for the generation of highly accurate data on multiple genes of hundreds of individuals in population-based studies. Two-dimensional gene scanning (TDGS) is a high-resolution system for detecting mutational variants in multiple genes in parallel. TDGS is based on a combination of extensive multiplex polymerase chain reaction (PCR) and two-dimensional (2-D) DNA electrophoresis. The latter involves a size separation step followed by denaturing gradient gel electrophoresis (DGGE). TDGS tests for a number of large human disease genes have been designed, using a computer program to optimally position PCR primers around the relevant target sequences (e.g., exons) and evaluated using panels of samples with previously detected mutations. The results indicate a high sensitivity and specificity, equal to nucleotide sequencing, which is generally considered as the gold standard. Here, we describe the different components of the TDGS process and its potential application as a high-throughput system for the systematic identification of human gene variants.

Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection.

The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. In this study, the nature of subtle lacZ mutations, which do not completely abolish beta-galactosidase activity, as detected by positive selection, was investigated. These subtle mutants are called 'color mutants' due to their light blue staining on X-gal medium. Replating of color mutants and retransformation of plasmid DNA, purified from individual color mutants, resulted in the same phenotype as the original color mutant. The p-gal positive selection system tolerates approximately 10% of wild-type activity as indicated by spectrophotometric determination of beta-galactosidase activity of individual color mutants. Restriction digestion and size separation of plasmid DNA revealed no visible change in the size of the plasmid in color mutants. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. In addition, it was discovered that the mouse line studied harbors four polymorphic base changes among the integrated plasmid copies.

Cell-by-cell scanning of whole mitochondrial genomes in aged human heart reveals a significant fraction of myocytes with clonally expanded deletions.

Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.

Design and application of 2-D DGGE-based gene mutational scanning tests.

Currently, there is a need for practical, accurate and cost-efficient tests to comprehensively scan human genes for disease-related DNA sequence variation. Two-dimensional gene scanning (TDGS) is a parallel mutation detection system, based on a combination of extensive multiplex PCR amplification ('PCR megaplex') and two-dimensional (2-D) DNA electrophoresis. The latter comprises a size separation step followed by denaturing gradient gel electrophoresis (DGGE), and allows single base pair changes to be distinguished among multiple DNA fragments in parallel. Here, we describe the rapid design of TDGS tests and its application to mutation identification in several large human cancer genes.

Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model.

Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation detection based on the electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. The use of denaturing gradient gel electrophoresis (DGGE) as the second separation step provides virtually 100% sensitivity, while the 2-D format allows the inspection of multiple gene fragments simultaneously. Analysis of many exons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has been applied to detect mutations in the retinoblastoma tumor suppressor gene RB1. Using RB1 as a model, we have now analyzed each step of the protocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An overall picture of TDGS cost-performance, as compared to direct sequencing, is provided as a function of the number of target fragments.

Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis.

An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutations by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost the entire mitochondrial genome, which then serves as template for the amplification of 25 short PCR fragments in two multiplex groups corresponding to regions implicated in human diseases. The mixture of fragments was subsequently subjected to two-dimensional electrophoretic separation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamide gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differences in melting behavior of mutant and wildtype fragments. Evaluation of the method using samples with known homoplasmic and heteroplasmic mutations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously identified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In addition, two variants were found to be novel mutations when analyzed by sequence analysis. One of these novel mutations was a heteroplasmic mutation in the COXIII gene that was found to segregate to homoplasmy in the next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected.

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.

Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests.

With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.

Accurate, high-throughput "snapshot" detection of hMLH1 mutations by two-dimensional DNA electrophoresis.

The human genome and related projects have resulted in the isolation of a rapidly growing number of genes that cause susceptibility to human cancer. With rare exception, these genes are large and have disease-associated mutations scattered along the length of the genes. Therefore, the development of accurate and cost-efficient mutation detection tests that can scan entire genes singly or in combination is warranted. hMLH1, encoding a mismatch repair enzyme, is a susceptibility gene for hereditary nonpolyposis colon cancer syndrome. This gene comprises 19 exons; mutations are scattered, typical of many susceptibility genes. Here, we present a strategy that combines extensive PCR multiplexing and two-dimensional DNA electrophoresis (Two-Dimensional Gene Scanning, TDGS) to scan accurately for mutations that lie within the exons and splice junctions of hMLH1. All target fragments, designed to have optimal melting characteristics, were prepared in a two-stage PCR--a four-plex long-distance PCR followed by short PCR in two multiplex groups of 10 and 11 amplicons. The mixture of amplicons was subjected to two-dimensional electrophoresis: separation by size in the first dimension and by melting characteristics in the second. Using this design, 41 samples containing known hMLH1 sequence variants or no alterations were blindly subjected to TDGS. All mutations were detected; there were no genuine false-positive or false-negative results. These results confirm that TDGS is a generally applicable, rapid, accurate, and reproducible mutation detection technology that would serve large-scale molecular epidemiologic studies as well as clinical molecular diagnostic purposes.

Exclusion of PTEN and 10q22-24 as the susceptibility locus for juvenile polyposis syndrome.

Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.

Denaturing gradient gel electrophoresis (DGGE) increases resolution and informativity of Alu-directed inter-repeat PCR.

By inter-repeat PCR, multiple polymorphic loci can be targeted in parallel. To improve resolution and extend the number of detectable polymorphisms, Alu-directed inter-repeat PCR products from two large pedigrees of the Centre d'Etude du Polymorphisme Humain (CEPH) were electrophoretically resolved in non-denaturing polyacrylamide gels and, separately, on the basis of sequence content by denaturing gradient gel electrophoresis (DGGE). The resolution in DGGE gels was found to be superior to that in non-denaturing gels and a higher number of fragments was detected separately. The number of polymorphic bands detected by DGGE alone, however, was lower than that after size separation. This is ascribed to the fact that because of complete melting, small polymorphic fragments can run off the gel. With three Alu-specific primers, 18 and 16 polymorphic bands per individual were detected by size separation in pedigrees 1200 and 6600, respectively. In the same two pedigrees, seven and 15 polymorphic bands, respectively, were detected by DGGE. Segregation analysis of polymorphisms in the CEPH pedigrees indicated that most polymorphisms detected by DGGE were different from those detected by size separation.

Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.

With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations.