Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4

Background

Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simpliCity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation.

Results

SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe.

Conclusions

Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.     

Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.     

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.     

Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

Background  

In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature.     

Precision-mapping and statistical validation of quantitative trait loci by machine learning

Background

DNA sequence diversity within the human genome may be more greatly affected by copy number variations (CNVs) than single nucleotide polymorphisms (SNPs). Although the importance of CNVs in genome wide association studies (GWAS) is becoming widely accepted, the optimal methods for identifying these variants are still under evaluation. We have previously reported a comprehensive view of CNVs in the HapMap DNA collection using high density 500 K EA (Early Access) SNP genotyping arrays which revealed greater than 1,000 CNVs ranging in size from 1 kb to over 3 Mb. Although the arrays used most commonly for GWAS predominantly interrogate SNPs, CNV identification and detection does not necessarily require the use of DNA probes centered on polymorphic nucleotides and may even be hindered by the dependence on a successful SNP genotyping assay.

Results

In this study, we have designed and evaluated a high density array predicated on the use of non-polymorphic oligonucleotide probes for CNV detection. This approach effectively uncouples copy number detection from SNP genotyping and thus has the potential to significantly improve probe coverage for genome-wide CNV identification. This array, in conjunction with PCR-based, complexity-reduced DNA target, queries over 1.3 M independent NspI restriction enzyme fragments in the 200 bp to 1100 bp size range, which is a several fold increase in marker density as compared to the 500 K EA array. In addition, a novel algorithm was developed and validated to extract CNV regions and boundaries.

Conclusion

Using a well-characterized pair of DNA samples, close to 200 CNVs were identified, of which nearly 50% appear novel yet were independently validated using quantitative PCR. The results indicate that non-polymorphic probes provide a robust approach for CNV identification, and the increasing precision of CNV boundary delineation should allow a more complete analysis of their genomic organization.     

Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

Background

Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.

Results

Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.

Conclusion

Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.     

Detection of single-nucleotide polymorphisms in the <Emphasis Type="Italic">p53</Emphasis> gene by LDR/RCA in hydrogel microarrays

To find single-nucleotide polymorphisms (SNPs) in the human genome, three modern technologies of molecular genetic analysis were combined: the ligase detection reaction (LDR), rolling circle amplification (RCA), and immobilized microarray of gel elements (IMAGE). SNPs were detected in target DNA by selective ligation of allele-specific nucleotides in microarrays. The ligation product was assayed in microarray gel pads by RCA. Two variants of microarray analysis were compared. One included selective ligation of short oligonu-cleotides immobilized in a microarray with subsequent amplification with a preformed circular probe (a common circle). The probe was especially designed for human genome research. The other variant employed immobilized allele-specific padlock probes, which could be circularized as a result of selective ligation. Codon 72 SNP of the human p53 gene was used as a model. RCA in microarrays proved to be a quantitative assay and, in combination with LDR, allowed efficient discrimination of alleles. The principles and prospects of LDR/RCA in microarrays are discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 30–39.Original Russian Text Copyright © 2005 by Kashkin, Strizhkov, Gryadunov, Surzhikov, Grechishnikova, Kreindlin, Chupeeva, Evseev, Turygin, Mirzabekov.     

滚环复制技术的建立及在RNA病毒基因检测中的初步应用

滚环复制是噬菌体繁殖所采取的一种基因复制方式,这种方式可使单链的环形分子在聚合酶和引物的作用下进行体外自我扩增。本文中用可特异性连接环化的寡核苷酸链作为探针,分别进行了1份细胞培养的禽流感病毒H5N1亚型样品、1份细胞培养的SARS病毒样品和4份丙型肝炎病毒阳性血清样品的检测。检测原理是探针与靶序列杂交后便可在T4DNA连接酶的作用下形成滚环复制中的环化单链分子,该分子在同温下可被特异性引物滚动复制和支链扩增。本文还利用按禽流感病毒NA1基因区序列合成的模拟DNA分子对该检测方法的灵敏度进行了测试。结果显示：利用固相RCA技术成功检测到三种RNA病毒的基因,该方法的灵敏度可达到能检测10^3拷贝模式DNA分子的水平。与传统的PCR方法敏感性的比较尚待进一步研究。     

基于PLP-LDR-HRCA策略进行微量DNA分析——rs17750303位点分型

增强PCR和全基因组扩增是当前微量DNA分析的主要策略,但是,由于DNA模板量过少,受随机效应影响显著,往往不能得到可靠的DNA分型结果.本文提出一种新的检验策略:PLP-LDR-HRCA,尝试微量DNA检材的SNPs分型研究.选择rs17750303位点,并设计等位基因特异性锁式探针,采用连接酶检测反应来识别等位基因,而后采用超分支滚环扩增反应来放大检测信号.结果表明,PLP-LDR-HRCA反应特异性好,灵敏度高,能够直接鉴别微量基因组DNA模板中待测SNP位点,rs17750303纯合型样品(AA型或CC型)和杂合型样品(AC型)准确分型所需最少模板量分别为20pg和30pg.对于增强PCR和全基因组扩增技术不能有效检验的微量检材,PLP-LDR-PCR策略独具优势,可能具有较大的开发价值.     

Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Background

Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray? genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.

Results

Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.

Conclusions

We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.     

<Emphasis Type="Italic">In situ</Emphasis>detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

Background  

In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification

In this review I will describe the development of a technique that enables genotyping of individual DNA molecules in the context of morphologically preserved fixed cells, from the fundamental concept published in 1994 to the present status. The review describes enzyme-assisted histochemistry approaches to achieve highly specific molecular identification reactions coupled to efficient signal amplification. The primary molecular identification is accomplished through circularization of oligonucleotide probes, called padlock probes. The circularization reaction is catalyzed by a DNA ligase, which provides robust distinction between single-nucleotide variants under standard reaction conditions. To generate a detectable signal from individual circularized probe molecules, a DNA polymerase is added that replicates probe circles, generating a long tandem-repeated DNA product, easily visualized using a standard epi-fluorescence microscope. Individual signals are recorded as bright dots, providing digital information about the abundance of specific sequences and opportunities for simultaneous detection of several targets using spectral multiplexing. The importance of strictly target-dependent signal amplification will be discussed.Robert Feulgen Prize 2006 Winner lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

Genotyping panel for assessing response to cancer chemotherapy

Background

Variants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability can result from genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc), and proteins relevant to cell survival (e.g., cell cycle, DNA repair, and apoptosis). The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and -sensitivity, and treatment outcomes.

Methods

We have adopted SNPlex for genotyping 432 single nucleotide polymorphisms (SNPs) in 160 candidate genes implicated in response to anticancer chemotherapy.

Results

The genotyping panels were applied to 39 patients with chronic lymphocytic leukemia undergoing flavopiridol chemotherapy, and 90 patients with colorectal cancer. 408 SNPs (94%) produced successful genotyping results. Additional genotyping methods were established for polymorphisms undetectable by SNPlex, including multiplexed SNaPshot for CYP2D6 SNPs, and PCR amplification with fluorescently labeled primers for the UGT1A1 promoter (TA)nTAA repeat polymorphism.

Conclusion

This genotyping panel is useful for supporting clinical anticancer drug trials to identify polymorphisms that contribute to interindividual variability in drug response. Availability of population genetic data across multiple studies has the potential to yield genetic biomarkers for optimizing anticancer therapy.     

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

Background

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.

Methodology/Principal Findings

The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G''s in their sequence.

Conclusions

The applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested.     

Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusariumspecies

Background

Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.

Results

The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.

Conclusion

The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.