M13 clones carrying point mutations: identification by solution hybridization

A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.     

Analysis of mutational spectra by denaturing capillary electrophoresis

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.     

Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).     

Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis

The identification of novel sequence variants, which may be either disease-causing mutations or silent polymorphisms, in large numbers of samples is becoming the rate-limiting step in associating diseases with specific genes. This is particularly true in light of the imminent arrival of the complete reference sequence of the human genome. A number of techniques have been developed to analyze DNA samples for sequence variants rapidly. We describe a new technique, capillary-based conformation-sensitive gel electrophoresis (capillary CSGE) that transfers mutation detection from acrylamide gel to capillary electrophoresis. Capillary CSGE was able to detect 7/7 short insertion/deletions and 16/22 base substitutions in a series of random single-nucleotide polymorphisms and known variants in the lipoprotein lipase and BRCA2 genes. This technique has the potential to screen many megabases of DNA in a single day.     

A human genome structural variation sequencing resource reveals insights into mutational mechanisms

Understanding the prevailing mutational mechanisms responsible for human genome structural variation requires uniformity in the discovery of allelic variants and precision in terms of breakpoint delineation. We develop a resource based on capillary end sequencing of 13.8 million fosmid clones from 17 human genomes and characterize the complete sequence of 1054 large structural variants corresponding to 589 deletions, 384 insertions, and 81 inversions. We analyze the 2081 breakpoint junctions and infer potential mechanism of origin. Three mechanisms account for the bulk of germline structural variation: microhomology-mediated processes involving short (2-20 bp) stretches of sequence (28%), nonallelic homologous recombination (22%), and L1 retrotransposition (19%). The high quality and long-range continuity of the sequence reveals more complex mutational mechanisms, including repeat-mediated inversions and gene conversion, that are most often missed by other methods, such as comparative genomic hybridization, single nucleotide polymorphism microarrays, and next-generation sequencing.     

High‐throughput microsatellite genotyping in ecology: improved accuracy,efficiency, standardization and success with low‐quantity and degraded DNA

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high‐throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR‐amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low‐quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.     

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3′ UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.     

A Method for Genome Comparisons and Hybridization Studies Using Known Megabase-Scale DNA Sequences as a Reference

We present a method for genome comparisons and high-resolution hybridization analyses using megabase stretches of known DNA sequences as a reference. The method employs two-dimensional gel electrophoresis, separating genomic segments cut with different restriction endonucleases in the first and second dimensions, to generate filters suitable for image analysis and repeated nucleic acid hybridizations. The corresponding two-dimensional pattern is computed from the reference nucleotide sequence and matched to the observed pattern, thereby identifying each fragment on the filter; at the same time the technique uncovers discrepancies from the reference sequence. This permits genome comparisons as well as automated identification and quantification of hybridization patterns with various probes. The technique is illustrated by an analysis ofSaccharomyces cerevisiaechromosome IX.     

Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes.

A method for detecting sequence variation of hypervariable segments of the mtDNA control region was developed. The technique uses hybridization of sequence-specific oligonucleotide (SSO) probes to DNA sequences that have been amplified by PCR. The nucleotide sequences of the two hypervariable segments of the mtDNA control region from 52 individuals were determined; these sequences were then used to define nine regions suitable for SSO typing. A total of 23 SSO probes were used to detect sequence variants at these nine regions in 525 individuals from five ethnic groups (African, Asian, Caucasian, Japanese, and Mexican). The SSO typing revealed an enormous amount of variability, with 274 mtDNA types observed among these 525 individuals and with diversity values, for each population, exceeding .95. For each of the nine mtDNA regions significant differences in the frequencies of sequence variants were observed between these five populations. The mtDNA SSO-typing system was successfully applied to a case involving individual identification of skeletal remains; the probability of a random match was approximately 0.7%. The potential useful applications of this mtDNA SSO-typing system thus include the analysis of individual identity as well as population genetic studies.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

Shotgun protein sequencing: assembly of peptide tandem mass spectra from mixtures of modified proteins

Despite significant advances in the identification of known proteins, the analysis of unknown proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for sequencing of unknown proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA sequencing strategies have not been extended to automated protein sequencing. We demonstrate, for the first time, the feasibility of automated shotgun protein sequencing of protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various proteins in western diamondback rattlesnake venom. We further argue that shotgun protein sequencing has the potential to overcome the limitations of current protein sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown proteins.     

Stabilizing Selection,Purifying Selection,and Mutational Bias in Finite Populations

Genomic traits such as codon usage and the lengths of noncoding sequences may be subject to stabilizing selection rather than purifying selection. Mutations affecting these traits are often biased in one direction. To investigate the potential role of stabilizing selection on genomic traits, the effects of mutational bias on the equilibrium value of a trait under stabilizing selection in a finite population were investigated, using two different mutational models. Numerical results were generated using a matrix method for calculating the probability distribution of variant frequencies at sites affecting the trait, as well as by Monte Carlo simulations. Analytical approximations were also derived, which provided useful insights into the numerical results. A novel conclusion is that the scaled intensity of selection acting on individual variants is nearly independent of the effective population size over a wide range of parameter space and is strongly determined by the logarithm of the mutational bias parameter. This is true even when there is a very small departure of the mean from the optimum, as is usually the case. This implies that studies of the frequency spectra of DNA sequence variants may be unable to distinguish between stabilizing and purifying selection. A similar investigation of purifying selection against deleterious mutations was also carried out. Contrary to previous suggestions, the scaled intensity of purifying selection with synergistic fitness effects is sensitive to population size, which is inconsistent with the general lack of sensitivity of codon usage to effective population size.     

Microchip electrophoresis: a method for high-speed SNP detection

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in ~100 s. This is 10 and 50 times faster than capillary and slab gel electrophoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only ±5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism^® 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.     

Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).

We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans

Constant denaturant electrophoresis is a DNA separation technique based on the principle of cooperative melting equilibrium. DNA sequences with distinct high and low melting domains can be utilized to separate and identify molecules differing by only one base pair in the lower melting domain. Combined with capillary gel electrophoresis and when coupled with high fidelity DNA amplification, this approach can detect mutants at a fraction of 10⁻⁶. Modifications to the capillary elecctrophoretic system have also increased DNA loading capacity which allows for analysis of rare mutations in a large, heterogeneous population such as DNA samples derived from human tissues. Employment of this technology has determined the first mutational spectrum in human cells and tissues in a mitochondrial sequence without phenotypic selection of mutants.     

MudPIT: multidimensional protein identification technology

Large-scale biology emerged out of the efforts to sequence genomes of important organisms. Based on resources created by whole genome sequencing, large-scale analyses of messenger RNA (mRNA) and protein expression are now possible. With the availability of large amounts of genomic sequence information, a convenient method for the identification and analysis of proteins based on proteolytic digestion into peptides emerged. Processes to fragment peptides using collision-activated dissociation (CAD) in tandem mass spectrometers and computer algorithms to match the tandem mass spectra of peptides to sequences in databases enable rapid identification of amino acid sequences, and hence proteins, present in mixtures. The inherent complexity of the peptide mixtures has necessitated improvements in methodology for mass spectrometry (MS) analysis of peptides.     

Viral discovery and sequence recovery using DNA microarrays

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.     

Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization

We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.