COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.     

DNA Sequences of Frameshift and Other Mutations Induced by Icr-170 in Yeast

ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs. However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.     

Estimating Long-Term Mating Systems Using DNA Sequences

Plant mating systems often involve a mixture of self fertilizations and outcross fertilizations. The degree of selfing has a large impact on the genetic composition of natural populations and on the evolution of the mating system itself in response to such factors as inbreeding depression. This paper describes a means of estimating the long-term rate of self-fertilization from samples of alleles taken from individuals in a population. Use is made of the genealogy of pairs of alleles at a locus within individuals and pairs between individuals. The degree of selfing is closely related to the extent to which the number of nucleotide sites differing within an individual is reduced relative to the number differing between individuals. Importantly, the estimate of long-term selfing is largely independent of population size and is not affected by historical fluctuations in population size; instead it responds directly to the mating system itself. The approach outlined here is most appropriate to evolutionary problems in which the long-term nature of the mating system is of interest, such as to determine the relationship between prior inbreeding and inbreeding depression.     

Estimating Effective Population Size or Mutation Rate Using the Frequencies of Mutations of Various Classes in a Sample of DNA Sequences

Mutations resulting in segregating sites of a sample of DNA sequences can be classified by size and type and the frequencies of mutations of different sizes and types can be inferred from the sample. A framework for estimating the essential parameter θ = 4Nu utilizing the frequencies of mutations of various sizes and types is developed in this paper, where N is the effective size of a population and μ is mutation rate per sequence per generation. The framework is a combination of coalescent theory, general linear model and Monte-Carlo integration, which leads to two new estimators θ(ξ) and θ(η) as well as a general Watterson''s estimator θ(K) and a general Tajima''s estimator θ(π). The greatest strength of the framework is that it can be used under a variety of population models. The properties of the framework and the four estimators θ(K), θ(π), θ(ξ) and θ(η) are investigated under three important population models: the neutral Wright-Fisher model, the neutral model with recombination and the neutral Wright''s finite-islands model. Under all these models, it is shown that θ(ξ) is the best estimator among the four even when recombination rate or migration rate has to be estimated. Under the neutral Wright-Fisher model, it is shown that the new estimator θ(ξ) has a variance close to a lower bound of variances of all unbiased estimators of θ which suggests that θ(ξ) is a very efficient estimator.     

犬科的线粒体细胞色素b DNA序列及其分子系统学研究

通过对犬科的赤狐、蓝狐、貉和狼４种的线粒体细胞色素ｂ约３７２ｂｐＤＮＡ片段序列分析,结合ＧｅｎＢａｎｋ中狗、西门豺和非洲野犬３种的该区段ＤＮＡ序列的比较,共发现１１３个核苷酸位点存在变异（约３０％）。ＮＪ法构建的分子系统树显示,非洲野犬最先从犬科动物中分化出来;犬属的我狼、狗和西门豺等３种为系统树上独立的一支,且其分歧的时间较赤狐、蓝狐和貉早;赤狐和蓝狐具有较近的亲缘关系。上述结果与形态的观点基本     

Non-Invasive Prenatal Diagnosis of Multiple Endocrine Neoplasia Type 2A Using COLD-PCR Combined with HRM Genotyping Analysis from Maternal Serum

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.     

Partition Enrichment of Nucleotide Sequences (PINS) - A Generally Applicable,Sequence Based Method for Enrichment of Complex DNA Samples

The dwindling cost of DNA sequencing is driving transformative changes in various biological disciplines including medicine, thus resulting in an increased need for routine sequencing. Preparation of samples suitable for sequencing is the starting point of any practical application, but enrichment of the target sequence over background DNA is often laborious and of limited sensitivity thereby limiting the usefulness of sequencing. The present paper describes a new method, Probability directed Isolation of Nucleic acid Sequences (PINS), for enrichment of DNA, enabling the sequencing of a large DNA region surrounding a small known sequence. A 275,000 fold enrichment of a target DNA sample containing integrated human papilloma virus is demonstrated. Specifically, a sample containing 0.0028 copies of target sequence per ng of total DNA was enriched to 786 copies per ng. The starting concentration of 0.0028 target copies per ng corresponds to one copy of target in a background of 100,000 complete human genomes. The enriched sample was subsequently amplified using rapid genome walking and the resulting DNA sequence revealed not only the sequence of a the truncated virus, but also 1026 base pairs 5′ and 50 base pairs 3′ to the integration site in chromosome 8. The demonstrated enrichment method is extremely sensitive and selective and requires only minimal knowledge of the sequence to be enriched and will therefore enable sequencing where the target concentration relative to background is too low to allow the use of other sample preparation methods or where significant parts of the target sequence is unknown.     

Estimation of DNA Sequence Context-dependent Mutation Rates Using Primate Genomic Sequences

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.     

用mtDNA序列鉴定一头小布氏鲸标本

测定了采自浙江省瑞安市的一头须鲸类标本的线粒体DNA（mitochondrial DNA,mtDNA)细胞色素b(cytochrome b,cyt b)基因369bp和控制区（control region)933bp的序列,通过与已发表的须鲸类同源序列比对,发现与西太平洋和日本水域的布氏鲸的cyt b基因和控制区分别有6.78%-7.05%和13.30%－14．40％的序列差异,而与来自所罗门群岛的布氏鲸之间cyt b基因的序列完全相同,控制区的序列也仅相差一个碱基（0.28%)。提示与邻近的西太平洋和日本海的普通布氏鲸在遗传上有显著区别,而可能与所罗门群岛的布氏鲸为同一种,即小布氏鲸（Balaenoptera edeni)。同时表明,应用分子生物学手段为进行鲸肉及其制品的种类鉴定是可行和有效的。     

用mtDNA控制区序列鉴定长须鲸标本

测定了采自长江口和杭州湾交汇海域的一头死亡大型须鲸骨骼标本的线粒体DNA(mt DNA)控制区序列(Control region)976 bp(登录号MF781125)、细胞色素C氧化酶Ⅰ基因(COⅠ)序列642 bp(登录号MG010134)和Cyt b序列307 bp(登录号MG010133)。通过与Gen Bank已发表的同源序列blast结果表明,与美国加利福尼亚海域长须鲸(Balaenoptera physalus)的控制区序列相似度达99%,仅在756 bp处有一个碱基T和C转换的差异;基于HKG+G模型,使用UPGMA聚类分析法和最大似然法(ML)构建的系统发育树与blast结果一致,故将标本鉴定为长须鲸,推测该个体来源于北太平洋。获取的细胞色素C氧化酶Ⅰ和Cyt b序列存在多个终止密码子,无法获取同源性较高的序列,推断可能为线粒体假基因。     

Multiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.     

应用基因芯片技术检测非综合征型耳聋基因突变

目的:应用遗传性耳聋基因芯片对散发性聋患者进行分子病因学检测,评估其在遗传性耳聋快速基因诊断中的可靠性。方法:门诊收集散发性聋患者10例,取外周血,提取基因组DNA,用遗传性耳聋基因芯片检测4个中国人中常见的耳聋相关基因中的9个热点突变,包括GJB2(35delG、176del16bp、235delC及299delAT)、GJB3(C538T)、SLC26A4(IVS7-2AG、A2168G)和线粒体DNA 12S rRNA(A1555G、C1494T)。同时,PCR扩增GJB2、线粒体12S rRNA基因全序列,DNA测序,以验证基因芯片检测结果的准确性。结果:在10名耳聋患者中,基因芯片方法检出1例携带线粒体DNA 12S rRNA C1494T突变;2例GJB2基因235delC纯合突变;2例235delC杂合突变;SLC26A4基因和GJB3基因未检出突变。基因芯片的结果与测序结果完全一致。结论:遗传性耳聋基因芯片技术对中国人常见耳聋相关基因热点突变的检出率高,结果准确、可靠,具有快速、高通量、高准确性、低成本等特点,能够满足临床耳聋基因检测的要求,同时结合产前诊断技术能有效预防耳聋患儿的出生,因而具有广阔的临床应用前景。     

利用多模板Y形接头延伸法进行相邻序列的连续扩增

Y形接头延伸法(Y-shaped adaptor dependent extension,YADE)是一种扩增已知DNA片段相邻序列的方法,但其效率常受到已知序列周围酶切位点数目的限制。在Y形接头延伸中采用由多种酶切和连接产生的DNA作模板,大大提高了该方法扩增相邻序列的效率,实现了相邻序列的连续扩增。利用该方法通过2轮连续的扩增从7种酶切连接产物中成功地获得了1个棉花小GTP酶基因(GhRacB)的2228bp上游序列。结果表明,多模板Y形接头延伸法是一种从复杂基因组中扩增相邻序列的有用方法。     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

HAlign 3: Fast Multiple Alignment of Ultra-Large Numbers of Similar DNA/RNA Sequences

HAlign is a cross-platform program that performs multiple sequence alignments based on the center star strategy. Here we present two major updates of HAlign 3, which helped improve the time efficiency and the alignment quality, and made HAlign 3 a specialized program to process ultra-large numbers of similar DNA/RNA sequences, such as closely related viral or prokaryotic genomes. HAlign 3 can be easily installed via the Anaconda and Java release package on macOS, Linux, Windows subsystem for Linux, and Windows systems, and the source code is available on GitHub (https://github.com/malabz/HAlign-3).