Novel multiplexed genotyping of human papillomavirus using a VeraCode-allele-specific primer extension method

A VeraCode‐allele‐specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)‐DNA. Oligonucleotide primers containing HPV‐type‐specific L1 sequences were annealed to HPV‐DNA amplified by PGMY‐PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin‐conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode‐ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY‐reverse blot hybridization assay, providing a new platform for high‐throughput genotyping required for HPV epidemiological surveys.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

A simple method to score single nucleotide polymorphisms based on allele‐specific PCR and primer‐induced fragment‐length variation

We describe a simple protocol to genotype single nucleotide polymorphisms (SNPs), which combines allele‐specific polymerase chain reaction (PCR) with fragment‐length analysis. Three primers are used in the PCR: two allele‐specific forward primers with a length‐difference and one reverse primer. The forward primers induce a length‐difference between the SNP‐variants, which can be assessed with standard fragment‐length analyses. We designed primers for 21 SNPs, and codominance was achieved for 76% of these SNPs. An inexpensive and flexible laser‐detection scoring protocol can be achieved with multiplex scoring and by incorporating the M13(‐21) genotyping method.     

Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II

J.‐H. Lee, N.‐W. Lee, S.‐W. Hong, Y.‐S. Nam, J.‐W. Choi and Y.‐S. Kim Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II Objective: To establish an efficient multiplex real‐time PCR assay for 15 human papillomavirus (HPV) genotypes, we designed multiplexing parameters and compared our PCR system with the hybrid capture (HC) II test using cervical cytology samples. Methods: For preventing cross‐reactive amplifications, variable HPV genes (E1, E2, E6, E7 and L1) were targeted. The melting temperatures of all primers and probes, and the size of the PCR product were optimized for the multiplex PCR. Our PCR system was compared with the HC II assays in the detection and genotyping of HPV infection using 173 cytology smears. Discordant cases between the two assays were verified by direct HPV DNA sequencing. Results: Of 173 women, 93 (53.8%) were HPV‐positive by the HC II assay and/or the multiplex real‐time PCR assay. The HPV genotypes were determined in 92 (98.9%) of 93 cases by the multiplex real‐time PCR and/or DNA sequencing. The agreement rate between multiplex PCR and HC II methods was 91.9% (kappa = 0.84). Although the sample size of this study needs to be increased to have epidemiological significance, multiple infections and HPV 16 were the predominant type. HPV 58, 52 and 18 accounted for 25% of HPV infections. HPV 52, 58 and 31 constituted 30% of CIN 2/3. Conclusion: The multiplex real‐time PCR system shows a good and reliable clinical performance. This in house PCR assay is fast and cost‐effective for HPV genotyping and the detection of HPV co‐infection in the post‐HPV vaccination era.     

Integrated allele-specific polymerase chain reaction-capillary electrophoresis microdevice for single nucleotide polymorphism genotyping

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

Two-temperature LATE-PCR endpoint genotyping

Background  

In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.     

Multiplex genotyping and allele frequency estimation in pooled DNAs using non-gel capillary electrophoresis

We present a novel approach of single-nucleotide polymorphism (SNP) analysis in which allele-specific oligonucleotide hybridization is followed by non-gel capillary electrophoresis (ASOH-NGCE) in conjunction with laser-induced fluorescence (LIF). This allows rapid multiplex allelotyping and allele frequency estimation. This method, based on site separation of the hybridization duplexes, retains the simplicity and specificity of ASOH and the homogeneous feature of NGCE with poly(N,N-dimethylacrylamide) (PDMA) as a sieving medium. ASOH-NGCE can be applied to multiplex SNP loci genotyping with excellent separation of hybridization mixtures. Average relative standard deviations (RSDs) were low for within-day (1.10%) and between-day (2.41%) reproducibility. Moreover, the allele frequencies in pooled DNAs were accurately determined from peak areas and equilibrium dissociation constants. Our method was highly sensitive in detecting alleles with frequency as low as 1% and in distinguishing allele frequencies differing by 1% between pools. The average value of differences between real and estimated frequencies (accuracy) was only 0.004.     

双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.     

Development of multiplex DNA electronic microarrays using a universal adaptor system for detection of single nucleotide polymorphisms

The NanoChip electronic microarray is designed for the rapid detection of genetic variation in research and clinical diagnosis. We have developed a multiplex electronic microarray assay, specific for single nucleotide polymorphism (SNP) genotyping and mutation detection, using universal adaptor sequences tailed to the 5' end of PCR primers specific to each target. PCR products, amplified by primers directed to the universal adaptor sequence, are immobilized on the microarray either directly or via capture oligonucleotides complementary to the universal adaptor sequence. This simple modification results in a significant increase in fidelity with improved specificity and accuracy. In addition, the multiplexing of genetic variant detection allows increased throughput and significantly reduced cost per assay. This general schema can also be applied to other microarray and macroarray formats.     

MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays

We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5′ end followed by locus-specific sequence at the 3′ end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of ~95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of ~75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Allele-specific PCR amplification due to sequence identity between a PCR primer and an amplicon : is direct sequencing so reliable?

PCR-direct sequencing (DS) is thought to be a very reliable method of determining DNA sequence and genotyping. Under certain conditions, however, DS can generate inaccurate results. Here we report a case of erroneous DS, in which a single nucleotide polymorphism (SNP) in the human PAX9 gene was mistyped due to allele-dependent PCR amplification. Examination of the amplified region showed that the 5' eight bases of one of the PCR primers were identical to the eight bases of the reverse strand downstream of the SNP, and the ninth base matched one of the alleles. Altering the primer so that it matched the other allele reversed the allele-specific inhibition. Reducing the base-pairing abolished the inhibition. Thus, the SNP was responsible for the difference in annealing efficacy of the primer and was therefore critical for the allele dependency. The allele-specific inhibition presented here can occur with any PCR primer sequence that encompasses a site that is polymorphic in the gene sequence. This phenomenon needs to be considered as a possibility when interpreting results from all PCR-based experiments. Sequence similarity between PCR primers and internal amplified regions should be considered for all methods for mutation detection and genotyping using PCR.     

Developing multiplexed SNP assays with special reference to degraded DNA templates

This protocol describes a single nucleotide polymorphism (SNP) genotyping strategy for highly degraded DNA, using a two-stage multiplex whereby multiple fragments are first amplified in a single exponential reaction and the products of this PCR are added to a linear single-base-extension reaction. It utilizes the analytical power of a capillary electrophoresis system to simultaneously type all the target sites. The protocol is specifically written for use with severely fragmented templates, typical of ancient DNA, and can be adapted to widely used detection platforms. The addition of the single-phase genotyping step avoids the need for the re-amplification and cloning of PCR products, while providing its own controls for the detection of contamination and allelic drop-out. This protocol can facilitate the routine analysis of up to 52 SNP markers (haploid or diploid) in 96 samples in a single day, and is recommended for the authentication of data in all areas of DNA research (population and medical genetics, forensics, ancient DNA).     

多重PCR同时检测人乳头瘤病毒、巨细胞病毒和沙眼衣原体

为了应用聚合酶链反应同时检测人巨细胞病毒（CMV）、人乳头瘤病毒（HPV）、沙眼衣原体（CT）,参照文献报道的基因序列,设计合成了三对能扩增370bp、450bp、510bp基因片段的引物,并对PCR扩增条件进行了优化。0.1fgHPV－DNA、CMV－DNA、CT－DNA即可被检出,得到了与设计片段相同的产物,且不扩增大肠杆菌、白色念球菌、解尿支原体等病原的核酸。对395例标本进行检查,各病原体的检出率分别是：HPV19.2%、CMV14.9%、CT5.1%。其中混合感染42例。PCR同时检测CMV、HPV、CT经济、快速、敏感、特异,可用于临床诊断和实验研究。     

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.     

Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system

We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5′-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.