LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E

Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.     

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of β-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the β-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.     

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel procedure for genotyping of single nucleotide polymorphisms in trisomy with genomic DNA and the invader assay

Individuals with trisomy 21 display complex phenotypes with differing degrees of severity. Numerous reliable methods have been established to diagnose the initial trisomy in these patients, but the identification and characterization of the genetic basis of the phenotypic variation in individuals with trisomy remains challenging. To date, methods that can accurately determine genotypes in trisomic DNA samples are expensive, require specialized equipment and complicated analyses. Here we report proof-of-concept results for an Invader® assay-based genotyping procedure that can determine SNP genotypes in trisomic genomic DNA samples in a simple and cost-effective manner. The procedure requires only two experimental steps: a real-time measurement of the fluorescent Invader® signal and analysis with a specifically designed clustering algorithm. The approach was tested using genomic DNA samples from 23 individuals with trisomy 21, and results were compared to genotypes previously determined with pyrosequencing. Additional assays for 15 SNPs were tested in a set of 21 DNA samples to assess assay performance. Our method successfully identified the correct SNP genotypes for the trisomic genomic DNA samples tested, and thus provides an alternative to determine SNP genotypes in trisomic DNA samples for subsequent association studies in patients with Down syndrome and other trisomies.     

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.     

Utility and accuracy of template-directed dye-terminator incorporation with fluorescence-polarization detection for genotyping single nucleotide polymorphisms

There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization detection (FP-TDI), in a sample of 177 SNPs selected solely on the basis of map location. Genotypes were generated without optimization using standard protocols. Overall, 81% of the SNPs we studied generated readable genotypes by FP-TDI. Thirty-two SNPs were genotyped in duplicate by PCR-RFLP orfluorescent dye-terminator sequencing. Out of a total of 631 duplicate genotypes, no true discrepancies were detected. The true error rate has a 95% chance of lying between 0 and 6 out of 1000 genotypes. We also tested for deviations from Hardy-Weinberg Equilibrium in 33 SNPs genotyped in 50 unrelated individuals, and no significant deviations were detected. Our FP-TDI data were readily adaptable to automated genotype calling using our own method of cluster analysis, which assigns a probability score to each genotype call. We conclude that FP-TDI is both efficient and accurate. The method can easily fill the needs of SNP genotyping projects at the scale typically used for regional or candidate-gene association studies.     

Capillary electrophoresis with laser-induced fluorescence detection for fast and reliable apolipoprotein E genotyping

The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 microM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping.     

Early kinetics of human macrophage apolipoprotein E synthesis and incorporation of carbohydrate precursors

Apolipoprotein E was detected in human macrophage lysates after 4-120-min pulses with [35S]methionine. Significant levels of apolipoprotein E were not found in the media until 120 min. Incorporation of N-[3H]acetylmannosamine, a sialic acid precursor, into apolipoprotein E was measurable after 120 min, whereas no [3H]mannose incorporation could be observed. These methods will be useful for examining the regulation of apolipoprotein E secretion and carbohydrate processing.     

A reexamination of the nucleotide incorporation fidelity of DNA polymerases

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.     

Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping.

Fluorescent hybridization probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent hybridization of 3'-fluorescein-labeled "donor" probes paired with 5'-labeled "acceptor" probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization probes of the same length, but different GC content are T(m) shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1 degrees C/s) annealing phase. Although each site preferred different concentrations of Mg(2+) and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.     

A novel procedure for efficient genotyping of single nucleotide polymorphisms

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of α-S-dNTPs and α-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5′-phosphodiesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the ‘GOOD Assay’ for SNP analysis.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Whole-genome genotyping with the single-base extension assay

We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.     

A monomeric human apolipoprotein E carboxyl-terminal domain

ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family. In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation. The aggregation property presents a major difficulty for the structural determination of this protein. A high-level expression system of the apoE C-terminal domain is reported here. Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant. This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E). The solubility of the mutant is significantly increased ( approximately 10-fold). Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL. CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein. DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain. In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins. These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity. Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.     

Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis

Isolated-probe PCR (IP-PCR) is a method that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting while maintaining a closed tube system. A double-stranded DNA (dsDNA) dye LCGreen I was used to detect the unlabeled probes. LCGreen I is also used to detect the 277-base pair PCR product peak as an internal amplification control. To accomplish this, IP-PCR separates the asymmetric PCR amplification step and the detection step of the unlabeled probes. This prevents the probes from interfering with the amplification of the DNA target. The samples are then melted using a high-resolution DNA melting instrument: the HR-1. The closed tube system virtually eliminates PCR product contamination or sample carryover The target apolipoprotein E (APOE) was chosen to test the IP-PCR technique. APOE contains two single nucleotide polymorphisms (SNPs) located 139 base pairs apart in a GC-rich region of the human genome. The results from this study show that the IP-PCR technique was able to determine the correct APOE genotype for each of the 101 samples. The IP-PCR technique should also be useful in detecting SNPs in other high-GC regions of the human genome.