The use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene

We describe the use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene. DNA fragments that differ in sequence by only a single base pair can be separated on denaturing gradient gels due to changes in their melting behavior. Previous studies have demonstrated the use of denaturing gradient gels to detect sequence changes in human genomic DNA, including mutations in the beta globin gene and polymorphisms on chromosome 20. We have begun to use denaturing gradient gels to look for polymorphisms within the human factor VIII gene. The DNA sequences of seven cloned fragments from introns in the human factor VIII gene were determined and used to predict a melting map for each fragment. The melting behavior of each cloned fragment was evaluated by electrophoresis into denaturing gradient gels. Appropriate fragments were then used as radioactive probes for hybridization to human DNA samples that had been digested with restriction enzymes. Heteroduplexes formed between the probe and genomic DNA samples were electrophoresed into denaturing gradient gels. The final positions of heteroduplex bands were determined by autoradiography. We describe a general approach for using denaturing gradient gel electrophoresis to find DNA polymorphisms, with particular emphasis on the predictive value of DNA sequence data. We compare the efficiency of polymorphism detection by denaturing gradient gel electrophoresis with detection by restriction fragment length polymorphism (RFLP) analysis. The factor VIII gene appears to have a low level of DNA sequence polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Notl-Msell methylation-sensitive amplied fragment length polymorhism for DNA methylation analysis of human cancers

We have applied a methylation-sensitive restriction endonuclease, NotI, to the existing amplified fragment length polymorphism (AFLP) method and developed NotI-MseI methylation-sensitive-AFLP (MS-AFLP). NotI-MseI MS-AFLP allows the analysis of DNA methylation alterations at the NotI sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel-eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent NotI-MseI MS-AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using NotI-MseI MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.     

Anion-exchange chromatography of DNA restriction fragments.

The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied. The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1. The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range. Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest. The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved. Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing. Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.     

Scanning the beta-globin gene for mutations in large populations by denaturing capillary and gel electrophoresis

Separation of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of 48 individual DNA samples. Four point mutations ranging in mutant frequency from 0.5 to 0.0002 were identified. Using CDCE with a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from 0.13 to 0.0005. This study shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in pangenomic scans to discover genes carrying casual mutations for common diseases.     

Species-specific polymerase chain reaction amplification of camel (Camelus) DNA extracts

A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.     

Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.     

Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography

Ion pair reverse phase high performance liquid chromatography on non-porous alkylated poly(styrene-divinylbenzene) particles enables the high resolution separation of double stranded DNA fragments. To further understand the separation mechanisms involved in ion pair reverse phase liquid chromatography we have analysed the effects of curved or "bent" DNA fragments with respect to their separation using both gel electrophoresis and ion pair reverse phase liquid chromatography. Size dependent separations of curved DNA fragments that migrate anomalously during gel electrophoresis were observed using ion pair reverse phase liquid chromatography. To further study the sequence effect and resulting changes in hydrophobicity of the duplex DNA, PCR fragments were generated that contain uracil in place of thymine. The resulting fragments were shown to elute with shorter retention times, demonstrating that sequence-specific effects can alter the retention of duplex DNA. The study was extended to the investigation of non-canonical B-DNA structures (Holliday junctions) under various chromatographic conditions, demonstrating that the coaxial stacking of the helices in such structures, in the presence of magnesium causes a change in retention.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Single-molecule immunoassay and DNA diagnosis

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.     

PCR/SSCP detects reliably and efficiently DNA sequence variations in large scale screening projects

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. for clarifying the genetic background of individuals with multifactorial diseases. Here we review our experience with the polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis to identify MPSV in a number of genes thought to be involved in the pathogenesis of multifactorial neurological disorders, including autoimmune diseases like multiple sclerosis (MS) and neurodegenerative disorders like Parkinson s disease (PD). The method is based on the property of the DNA that the electrophoretic mobility of single stranded nucleic acids depends not only on their size but also on their sequence. The target sequences were amplified, digested into fragments ranging from 50-240 base pairs (bp), heat-denatured and analysed on native polyacrylamide (PAA) gels of different composition. The analysis of a great number of different PCR products demonstrates that the detection rate of MPSV depends on the fragment lengths, the temperature during electrophoresis and the composition of the gel. In general, the detection of MPSV is neither influenced by their location within the DNA fragment nor by the type of substitution, i.e., transitions or transversions. The standard PCR/SSCP system described here provides high reliability and detection rates. It allows the efficient analysis of a large number of DNA samples and many different genes.     

An efficient unnatural base pair for PCR amplification

Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was approximately 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.     

A Novel Affinity Capillary Electrophoresis for the Detection of Gene Mutation Using Immobilized Oligonucleotides-Polyacrylamide Conjugate

It has been found that small mutations of certain genes are the definitive origin of many heritable disorders and cancers with striking development of recent molecular biology. Such new findings have taken close-up of the importance of gene mutation assays based on the difference of DNA base sequences in diagnostic or medical field Capillary electrophoresis can be a good candidate for an ideal method on such gene analysis, because the methods can be performed with trace amount of samples, high resolution and shorter running time. We have established an effect of oligonucleotide, which was introduced onto capillary inner surface, on the recognition of an overall sequence of sample DNA fragments as an affinity ligand.     

Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.     

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.     

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.     

Separation of long DNA fragments by inversion field capillary electrophoresis

This study reports improved pulsed field capillary electrophoresis (PFCE) for separation of large DNA ladders. Important analytical conditions, including gel polymer concentration, ratio of forward to backward pulse duration, and separation potential, were investigated for their effects on the separation performance of DNA ranging in size from 0.1 to 10.0 kilo base pairs (kbp). Results show that DNA fragments from 0.1 to 8.0 kbp can be resolved with high resolution, simultaneously, in a short time. The ratio of forward to backward pulse duration affects the separation performance for DNA fragments greater than 1.5 kbp, and 3 or 4 is the optimum value of the ratio for separation of DNA up to 10 kbp. Furthermore, the separations that were obtained with 74–19,329 bp λ-DNA restriction fragments clearly demonstrate a dramatic improvement in the separation time and resolution over the conventionally used square-wave PFCE. The inversion field capillary electrophoresis reported here may help enable future DNA analysis studies to be performed quickly and effectively.     

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).     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.