Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification Process.

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.     

四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基引入了一个人为错配碱基 ,使引物的错误延伸率显著降低 ,大大提高了SNP分析的准确性 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .     

Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

DNA sequencing has markedly changed the nature of biomedical research, identifying millions of polymorphisms along the human genome that now require further analysis to study the genetic basis of human diseases. Among the DNA-sequencing platforms available, Pyrosequencing has become a useful tool for medium-throughput single nucleotide polymorphism (SNP) genotyping, mutation detection, copy-number studies and DNA methylation analysis. Its 96-well genotyping format allows reliable results to be obtained at reasonable costs in a few minutes. However, a specific biotinylated primer is usually required for each SNP under study to allow the capture of single-stranded DNA template for the Pyrosequencing assay. Here, we present an alternative to the standard labeling of PCR products for analysis by Pyrosequencing that circumvents the requirement of specific biotinylated primers for each SNP of interest. This protocol uses a single biotinylated primer that is simultaneously incorporated into all M13-tagged PCR products during the amplification reaction. The protocol covers all steps from the PCR amplification and capture of single-stranded template, its preparation, and the Pyrosequencing assay itself. Once the correct primer stoichiometry has been determined, the assay takes around 2 h for PCR amplification, followed by 15-20 min (per plate) to obtain the genotypes.     

Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue

When we placed an ENA residue into primers at the 3' end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer-dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS-PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.     

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.     

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.     

High-level multiplex DNA amplification.

We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.     

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.     

基于荧光定量PCR扩增反应的SNP测定法

建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.     

The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.     

Tail variation of the folding primer affects the SmartAmp2 process differently

Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either “strong” or “weak”, depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with “strong tails”. Unlike “strong tails”, concentration change of FP with “weak tails” did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.     

Modified Proofreading PCR for Detection of Point Mutations,Insertions and Deletions Using a ddNTP-Blocked Primer

The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3''-5'' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3''-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 10² copies and a selectivity of 5 × 10^-5 mutant among 10⁷ copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach.     

Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons

Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP.     

一种DNA扩增的新技术： 利用热稳定的Bst DNA聚合酶驱动跨越式滚环等温扩增反应

Bst DNA聚合酶具有热稳定性、链置换活性及聚合酶活性,在体外DNA等温扩增反应中起重要作用. 本文利用Bst DNA聚合酶的5′→3′聚合酶、核苷酸（末端）转移酶及链置换酶活性发展了一种新的体外环式DNA扩增技术跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)．在SRCA反应中,Bst DNA聚合酶以上游引物P1为模板合成其互补链RcP1,并和P1形成双链DNA|之后,Bst DNA聚合酶用其核苷酸转移酶活性在其P1的3′末端沿5′→3′方向随机掺入脱氧核糖核苷酸聚合形成寡聚核苷酸（dNMP)m序列,即DNA的合成反应跨越了RcP1 与下游引物P2之间的缺口|然后,以下游引物P2为模板形成互补序列（RcP2）;接着,Bst DNA聚合酶继续将脱氧核糖核苷酸随机添加到RcP2的3′末端,形成（dNMP)n序列．继而,Bst DNA聚合酶以RcP1为模板,继续催化聚合反应合成互补新链,并通过其链置换酶活性替换P1|如此往复,形成［P1-(dNMP)m-RcP2-(dNMP)n …］序列．本文通过电泳、酶切、测序等方法对扩增产物进行分析,演绎出上述扩增过程,并就工作原理进行了讨论．该反应可能对开发等温扩增技术检测微生物有一定助益,也为解释环介导等温扩增技术中假阳性反应和滚环等温扩增反应中的背景信号提供了线索．     

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.     

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.     

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome.     

Detection of Fusarium wilt pathogens of Psidium guajava L. in soil using culture independent PCR (ciPCR)

Traditional culturing methods take a long time for identification of pathogenic isolates. A protocol has been developed for the detection of Fusarium from soil samples in the early stage of infection. Seventeen soil samples from different locations were collected before the onset of rains to find out the presence of Fusarium spp. population present in the soil of guava orchards and to correlate its presence with incidence of wilt. A PCR based method was developed for the molecular characterization of Fusarium using Fusarium spp. specific primer. DNA extracted by this method was free from protein and other contaminations and the yield was sufficient for PCR amplification. The primer developed in this study was amplifying ∼230 bp in all infected samples while not in healthy soil. The specificity and sensitivity of primer were tested on several Fusarium spp. and found that this primer was amplifying 10⁻⁶ dilution of the fungal DNA. The present study facilitates the rapid detection of Fusarium spp. from infected soil samples of guava collected from different agroclimatic regions in India. A rapid detection method for pathogens and a diagnostic assay for disease would facilitate an early detection of pathogen and lead to more effective control strategies.     

Direct transduction of allele-specific primer extension into electrical signal using genetic field effect transistor

We have developed a genetic field effect transistor (FET) for single nucleotide polymorphism (SNP) genotyping, which is based on potentiometric detection of molecular recognition on the gate insulator. Here, we report direct transduction of allele-specific primer extension on the gate surface into electrical signal using the genetic FETs. This method is based on detection of intrinsic negative charges of polynucleotide synthesized by DNA polymerase. The charge density change at the gate surface could be monitored during primer extension reaction. Moreover, three different genotypes could be successfully distinguished without any labeling for target DNA by the use of the genetic FET in combination with allele-specific primer extension. The platform based on the genetic FETs is suitable for a simple, accurate and inexpensive system for SNP genotyping in clinical diagnostics.     

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis

Abstract A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name ' Anguillina coll '. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for ' A. coli '. The PCR was used to correctly identify DNA extracted from 43 isolates of ' A. coli ' from humans and pigs, whilst no product was produced from Escherichia coli , or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi . The amplification provided a rapid and simple means of identifying DNA from isolates of ' A. coli ', and could be used on boiled whole ' A. coli ' cells, with a detection limit equivalent to 2.5 × 10² cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.