Single Nucleotide Extension Research Articles

High‐throughput mutational screening for beta‐thalassemia by single‐nucleotide extension

In this work a high-throughput method based on the single-nucleotide extension (SNE) reaction and multicolour detection in a DNA sequencer was developed to screen for eight mutations in the human beta-globin gene: IVSI.110, cd39, IVSI.1, IVSI.6, IVSII.745, HbC, HbS and cd6. The method has been validated on a large number of samples for the two most common mutations causing beta-thalassemia in the Mediterranean area (IVSI.110 and cd39). The development of a high-throughput, fast and reliable method to assay beta-thalassemia mutations represents a significant improvement in molecular diagnosis of this disease. The multicolour detection and the use of multiple injections further enhances the throughput of mutational screening by the DNA sequencer and facilitates automated genotyping for routine molecular diagnostics.

Simultaneous Amplification, Detection, and Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene

Classic galactosemia is an autosomal recessive inherited error of galactose metabolism. It is caused by lack of galactose-1-phosphate uridyl transferase, an enzyme that is required to metabolize galactose-1-phosphate to uridine diphosphate galactose. The build up of galactose-1-phosphate is toxic at high levels and can damage the liver, brain, eyes, and other vital organs. Over 200 mutations have been identified in affected individuals. We describe an assay to identify nine target mutations or variants in the galactose-1-phosphate uridyl transferase gene, namely p.Q188R, p.S135L, p.K285N, p.L195P, p.T138M, p.Y209C, IVS2-2 A>G, p.L218L, and p.N314D. A single long-range PCR is followed by a multiplexed nucleotide extension assay (single nucleotide extension) and capillary electrophoresis to detect simultaneously all nine target mutations/variants. Fifty-four previously characterized samples (47 clinical samples and seven controls) gave a 100% concordance. We also report a nontarget novel mutation, p.L192X, and its profile using single nucleotide extension. This assay can complement the enzyme activity assay and identify familial mutations for testing additional family members.

Discriminatory Power, Typability, and Accuracy of Single Nucleotide Extension Microarrays

Despite great conceptual promise, the use of microarrays in typing approaches has not yet gained wide acceptance. The establishment of proper criteria for determining discriminatory power as well as typability and the accuracy of microarray data remains to be solved. Purely experimental estimations of these parameters would far exceed what is experimentally practical. We therefore used simulations in combination with experimental results in parameter estimations. Our assay was based on 26 single nucleotide polymorphisms (SNPs) identified in the Campylobacterjejuni Multi Locus Sequence Typing (MLST) database (http:///pubmist.org/campylobacter/). The SNPs were detected using a single nucleotide extension (SNE) typing microarray. Unknown isolates were assigned to the known sequence type(s) by calculating weighted sum of matches minus a weighted sum of mismatches between predicted and candidate genotype. The weights were set according to the Bayesian posterior probability of the SNP classification. These studies showed that any typing or profiling method based on binary data requires an accuracy of <2-3% error for each datapoint (in our case SNPs) to classify the isolates to the correct allelic profile in 90% of the cases. The classification error for our experimental data was 3.2% (after removing 5 high error SNPs). We therefore conclude that SNE microarrays are promising for future high-throughput typing of bacteria.

The Role of Specific Amino Acid Residues in the Active Site of Escherichia coli DNA Polymerase I on Translesion DNA Synthesis Across from and Past an N-2-Aminofluorene Adduct

Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. A central part of these studies is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template. To address the importance of polymerase architecture on replication across from this type of bulky DNA adduct, three active-site mutants of Escherichia coli DNA polymerase I (Klenow fragment) were used to study DNA synthesis on DNA modified with the carcinogen N-2-aminofluorene (AF). Running-start synthesis studies showed that full-length synthesis past the AF adduct was inhibited for all of the mutants, but that this inhibition was substantially less for the F762A mutant. Single nucleotide extension and steady-state kinetic experiments showed that the Y766S mutant displayed higher rates of insertion of each incorrect nucleotide relative to WT across from the dG-AF adduct. This effect was not observed for F762A or E710A mutants. Similar experiments that measured synthesis one nucleotide past the dG-AF adduct revealed an enhanced preference by the F762A mutant for dG opposite the T at this position. Finally, synthesis at the +1 and +2 positions was inhibited to a greater extent for the Y766S and E710A mutants compared with both the WT and F762A mutants. Taken together, this work is consistent with the model that polymerase geometry plays a crucial role in both the insertion and extension steps during replication across from bulky DNA lesions.

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

Nucleotide Excision Repair and Template-independent Addition by HIV-1 Reverse Transcriptase in the Presence of Nucleocapsid Protein

During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.

Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis

With the increasing availability of genetic information and its relationship to human diseases, there is a growing need in the medical diagnostic field for technologies that can proceed to the parallel genotyping of multiple markers. In this paper, we report the development of a new flexible microarray-based method that aims to be inexpensive, accurate, and adapted to routine analysis. The construction of the MICAM® (MICrosystem for Analysis in Medicine) DNA chip is based on the controlled electrosynthesis of a conducting polymer film bearing oligonucleotide probes on gold electrodes. First, accessible 3′OH-ends of grafted probes are directly used to conduct single template-dependent nucleotide extension reactions with fluorescence-labeled chain terminators. Then, the fluorescence of incorporated dideoxynucleotides on controls and probes of interest are recorded to assess base calling. Here, we present the development of the methodology to assign the genotype of TP53 (tumor protein p53) codon 72 polymorphism and its application to analysis of genomic DNA from cell lines and from human colorectal samples. The genotyping results obtained by minisequencing on the polypyrrole DNA chip were 100% concordant with data obtained by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Moreover, the developed probe array assay has been successfully applied to the detection of TP53 loss of heterozygosity.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform.

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced ‘quantitative’ C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.

MagSNiPer: A new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3′ terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10 −17 mol of template DNA.

Polymorphisms in XRCC1 Modify the Association between Polycyclic Aromatic Hydrocarbon-DNA Adducts, Cigarette Smoking, Dietary Antioxidants, and Breast Cancer Risk

The variability in DNA repair capacity of the general population may depend in part upon common variants in DNA repair genes. X-ray repair cross complementing group 1 (XRCC1) is an important DNA base excision repair gene and exhibits polymorphic variation. Using the Long Island Breast Cancer Study Project, a population-based case-control study, we evaluated the hypothesis that two common single nucleotide polymorphisms of XRCC1 (codon 194 Arg-->Trp and 399 Arg-->Gln) influence breast cancer susceptibility and interact with polycyclic aromatic hydrocarbon (PAH)-DNA adducts, cigarette smoking, and intake of fruits and vegetables and antioxidants. The available sample for genotyping included 1,067 cases and 1,110 controls. Genotyping was done by a high-throughput single-nucleotide extension assay with fluorescence polarization detection of the incorporated nucleotide. We observed no significant increases in risk among all subjects who were carriers of XRCC1 194Trp or 399Gln alleles. Among never smokers, we observed an increased risk of breast cancer in 399Gln carriers [odds ratio (OR), 1.3; 95% confidence interval (CI), 1.0-1.7). Further analysis indicated a suggestive weak additive interaction between the 399Gln allele and detectable PAH-DNA adducts (OR for exposure with mutant genotype, 1.9; 95% CI, 1.2-3.1). The estimated age-adjusted interaction contrast ratio (ICR) and 95% CI (ICR, 0.38; 95% CI, -0.32 to 1.10) indicated that the departure from additivity was not statistically significant, but that there was some suggestion of a relative excess risk due to the interaction. In subjects with at least one copy of XRCC1 194Trp allele, there was a moderate interaction with high intake of fruits and vegetables (>/=35 half-cup servings per week of any fruits, fruit juices, and vegetables, OR, 0.58; 95% CI, 0.38-0.89; ICR, -0.49; 95% CI, -0.03 to -0.95), and dietary plus supplement antioxidant intake with 33% to 42% decreases in breast cancer risk compared with those with the Arg194Arg genotype and low-intake individuals. These results do not show that the two genetic polymorphisms of XRCC1 independently influence breast cancer risk. However, there is evidence for interactions between the two XRCC1 single nucleotide polymorphisms and PAH-DNA adducts or fruit and vegetable and antioxidant intake on breast cancer risk. Further understanding of the biological function of XRCC1 variants and their interactions with PAH-DNA adducts, antioxidants, and other genes in the pathway are needed.

Accelerating chemical replication steps of RNA involving activated ribonucleotides and downstream-binding elements

Template-directed single nucleotide extension of an RNA primer with oxyazabenzotriazolides of ribonucleotides is shown to be fast and sequence-selective; downstream-binding RNA strands contribute to the acceleration of the reaction.

Allele level typing of HLA-B locus by multiplexed single nucleotide extension (MSNE) and a study of b15 in different ethnic groups

Allele level typing of HLA-B locus by multiplexed single nucleotide extension (MSNE) and a study of b15 in different ethnic groups

HLA class I genes in the SHE ethnic minority of South China determined by multiplexed single nucleotide extension (MSNE)

HLA class I genes in the SHE ethnic minority of South China determined by multiplexed single nucleotide extension (MSNE)

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

A Single Nucleotide Primer Extension Assay to Detect the APC I1307K Gene Variant

Adenomatous polyposis coli (APC) is a tumor suppressor gene important in colorectal tumorigenesis. A genetic variant of APC, I1307K, results from a T-to-A transversion at nucleotide 3920 which converts the wild-type sequence to a homopolymer tract (A(8)). The I1307K alteration is not itself oncogenic, but creates a hypermutable region (A(8)) that is prone to frame-shift mutations. The APC I1307K variant occurs in approximately 6% of the Ashkenazi Jewish population and is reported to approximately double an individual's risk for colorectal cancer. Here we describe a single nucleotide primer extension assay for the detection of the APC I1307K mutation. Following PCR amplification, nucleotide 3920 of the APC gene is directly sequenced using single nucleotide primer extension technology. The assay is in a multiplex format allowing simultaneous forward and reverse sequencing of the I1307K variant, which provides an internal, independent confirmation of each testing result. The assay was validated against 60 samples previously characterized by an allele-specific oligonucleotide (ASO) hybridization assay, with 100% concordance of results. Compared to the ASO assay, this single nucleotide primer extension assay requires significantly less technical time to perform, and has a greatly increased throughput capacity. The single nucleotide extension assay provides a highly sensitive and specific assay to identify individuals with the APC I1307K gene variant who may benefit from increased colorectal screening.

Comparison of GenFlex Tag Array and Pyrosequencing in SNP Genotyping

With the completion of the Human Genome Project, over 2 million sequence-verified single nucleotide polymorphisms (SNPs) have been deposited in public databases. The challenge has shifted from SNP identification to high-throughput SNP genotyping. Although this has had little impact on molecular diagnostics, it provides the potential for future molecular diagnostics of complex traits to include SNP profiling. Accordingly, efficient, accurate, and flexible SNP genotyping are needed. In addition, the drive for low cost has pushed genotyping reactions toward multiplexing capability. We compared two SNP genotyping techniques: Affymetrix GenFlex Tag array and Pyrosequencing. The reference method was a well-established, solid-phase, single nucleotide extension reaction technique based on tritium detection. Fourteen SNPs were selected from the fine mapping project of a multiple sclerosis locus on chromosome 17q. Using all three techniques and the reference method, the SNPs were analyzed in 96 related individuals. Without extensive optimization, we successfully genotyped 11 of 14 SNPs with both GenFlex and Pyrosequencing. Our study suggests that the Pyrosequencing technique provides higher accuracy between the two systems which is most likely due to the single-stranded template in the extension reaction. Thus, Pyrosequencing has potential for diagnostic applications. Pyrosequencing, however, is not optimal for large SNP profiling analyses wherein multiplexing potential is an advantage.

Typing for all known HLA-DR alleles by group-specific PCR and flow cytometry-based multiplexed single nucleotide extension

Typing for all known HLA-DR alleles by group-specific PCR and flow cytometry-based multiplexed single nucleotide extension