Optimal Primer Research Articles

Development of a rapid and high-throughput molecular method for detecting the F200Y mutant genotype in benzimidazole-resistant isolates of Fusarium asiaticum.

The point mutation at codon 200 (TTC→TAC, F200Y) of the β2 -tubulin gene confers resistance to benzimidazole fungicide in Fusarium asiaticum. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration (MIC) of fungicides, which is always time consuming, tedious and inefficient. A visual, rapid and efficient method with high specificity was developed, based on loop-mediated isothermal amplification (LAMP). Six sets of LAMP primers were designed, and one set was optimised specifically to distinguish the F200Y mutant genotype. With the optimal LAMP primers, concentrations of LAMP components were optimised. The optimal reaction conditions were 57-64 °C for 75 min. The feasibility of the LAMP assay for detection of the F200Y mutant genotype of F. asiaticum was demonstrated by assaying diseased wheat spikelets that were artificially inoculated in the field. The new LAMP assay had good specificity, sensitivity, stability and repeatability. It will be useful for assessing the risk of F. asiaticum populations with carbendazim resistance developing in the field, and will also provide important reference data for integrated control of Fusarium head blight caused by F. asiaticum. © 2016 Society of Chemical Industry.

A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR).

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was 65°C. The optimized template and primer concentration were 1.5 μL (50 ng/μL) and 3 μL (10 μM/μL) respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at 88.0°C, 87.5°C, 83.5°C, and 89.5°C respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60–64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.

오징어류 종 판별을 위한 다중 유전자 검사법 개발 및 검증

본 연구에서는 오징어류에 해당하는 대왕오징어, 오징어, 문어, 한치 및 이를 이용한 가공식품에 대해서 분자생물학적 기법을 활용한 시험법을 검토하였다. 시료 중 원료성분 확인을 위하여 오징어류 4종에 대해 최적의 종 특이 프라이머를 디자인하였으며, 시료로부터 직접 genomic DNA를 추출하여 PCR을 실시하였다. PCR 수행과정에서 반응을 저해하는 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR을 실시한 결과, Single PCR의 경우 대왕오징어(552 bp), 오징어(463 bp), 문어(247 bp), 한치(354 bp)에 해당하는 종 특이적인 증폭산물을 확인하였으며, Multiplex PCR 의 경우 서로 다른 종 사이의 교차반응없이 동시다발적으로 증폭이 일어남을 확인할 수 있었다. 또한 이들 4종에 대해 PCR 민감도를 조사한 결과, 모두 약 <TEX>$0.1ng/{\mu}l$</TEX>의 농도까지 검출이 가능함을 확인하였으며 multiplex PCR의 경우 약 <TEX>$0.25ng/{\mu}l$</TEX>의 농도까지 검출이 가능함을 확인하였다. 이를 이용하여 오징어류가 함유된 수산물 가공식품 8건에 대해 적용성을 검토한 결과, 모든 시료에서 유효한 결과를 확인할 수 있었다. 따라서 본 연구에서 제작된 오징어류 4종에 대한 종 특이적 프라이머는 생물 상태뿐만 아니라 수산물 가공식품에 대해서도 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다. In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.

Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation.

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2’s Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0134-8) contains supplementary material, which is available to authorized users.

Sequence-related amplified polymorphism primer screening on Chinese fir (Cunninghamia lanceolata (Lamb.) Hook)

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the most important coniferous tree species used for timber production in China. Here, we conducted a sequence-related amplified polymorphism (SRAP) primer screening assay with a total of 594 primer combinations, using 22 forward and 27 reverse primers on four representative Chinese fir genotypes. The obtained results indicated that Chinese fir genomic DNA has a notable amplification bias on the employed forward or reverse primer nucleotides (3′ selection bases). Out of the tested primer sets, 35 primer combinations with clearly distinguished bands, stable amplification, and rich polymorphism were selected and identified as optimal primer sets. These optimal primer pairs gave a total of 379 scorable bands, including 265 polymorphic bands, with an average of 10.8 bands and 7.6 polymorphic bands per primer combination. The produced band number for each optimal primer set ranged from 7 to 14 with a percentage of polymorphic bands spanning from 33.3 to 100.0 %. These primer combinations could facilitate the next SRAP analysis assays in Chinese fir.

Rapid and simple method of qPCR primer design.

Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.

MultiPLX: automatic grouping and evaluation of PCR primers.

In this chapter we describe MultiPLX-a tool for automatic grouping of PCR primers for multiplexed PCR. Both generic working principle and step-by-step practical procedures with examples are presented. MultiPLX performs grouping by calculating many important interaction levels between the different primer pairs and then distributes primer pairs to groups so that the strength of unwanted interactions is kept below user-defined compatibility level. In addition it can be used to select optimal primer pairs for multiplexing from list of candidates. MultiPLX can be downloaded from http://bioinfo.ut.ee/?page_id=167. Graphical web-based interface to most functions of MultiPLX is available at http://bioinfo.ut.ee/multiplx/.

From primer design to validation of results - is it possible by using free software only? / De la proiectarea primerilor la validarea rezultatelor - este posibil utilizând doar programe gratuite?

AbstractSuccessful experiments in molecular biology require good knowledge about various methods and protocols. In molecular biology, nucleic acid manipulation is the essence, starting with the quality of extraction and ending with several analysis assays (PCR, RT-PCR, qPCR, PCR arrays, molecular cloning, etc). Though many of these are so called “standardized”, in practice there are many variables that can influence the outcome of the experiment. Due to the importance of optimal primer design in PCR assays, we will focus on primer designing and checking software, but we also present other useful free tools that can help researchers in the molecular biology field

PrimerDesign-M: a multiple-alignment based multiple-primer design tool for walking across variable genomes.

Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. Furthermore, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions. PrimerDesign-M is available as a webtool at http://www.hiv.lanl.gov/content/sequence/PRIMER_DESIGN/primer_design.html tkl@lanl.gov or seq-info@lanl.gov.

A novel approach to prenatal fetal sex diagnosis by detecting an insertion in the Y-chromosome of ovine Amelogenin gene

It is completely clarified that both intact fetal cells and cell-free fetal nucleic acids cross the placenta and circulate in the maternal blood stream during pregnancy. In the current study we employed a new approach in applying Amelogenin gene for fetal sex prediction in ovine species. The amount of fetal DNA in the maternal blood was quantified using quantitative real-time PCR technique. Peripheral blood was obtained from 45 pregnant sheep with age of pregnancy varying from 8 to 18 weeks. Optimal primers were designed on Amelogenin intron 4, a Y specific insertion region in Amelogenin gene. For 42 (93.3%) ewes, agreement was verified between fetal sex prediction using cffDNA (cffDNA) and birth outcome. The test sensitivity, specificity and accuracy of fetal sex prediction were 96.5%, 87.5% and 93.3% respectively. The result of relative quantification showed that the cell-free fetal DNA values were significantly higher in ewes with more than 3 months of pregnancy relative to those with less than it. In conclusion, an insertion part in the ovine Y-chromosome Amelogenin gene could be successfully used for fetal sex prediction in ovine species using cell-free fetal DNA.

A Rapid Multiplex PCR‐DHPLC Method of Detection and Identification of Pathogenic Bacteria in Aquatic Products

AbstractIn this study, we established a multiplex polymerase chain reaction‐denaturing high‐performance liquid chromatography (MPCR‐DHPLC) method for rapid detection of the aquatic‐associated pathogens Vibrio cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and Listeria monocytogenes. Specific primer sets targeting the dnaJ gene of the Vibrio species and the hly gene of L. monocytogenes were used. We also defined the optimal primer concentrations to detect each Vibrio species. We used the MPCR‐DHPLC method to identify target bacteria in aquatic products. Performance characteristics of the MPCR‐DHPLC system showed that it is specific for six common pathogenic bacterial species and can be used for the practical detection of these pathogens in aquatic products. Furthermore, two practical applications of two environmental water samples were tested according to this method by MPCR‐DHPLC, then the results of detection were consistent with conventional tests but faster. It showed that this method proved to be a fast, sensitive and specific tool for V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and L. monocytogenes detection in a routine microbiological laboratory.Practical ApplicationsVarious bacterial species can be simultaneously detected with the same PCR reaction. A MPCR‐DHPLC method of identification of various bacterial species was established. This method improves test efficiency and minimizes test time.

Sensitive and Rapid Detection of ermB Gene in Clostridium difficile by Loop-Mediated Isothermal Amplification

Introduction: Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. This paper describes a sensitive and rapid molecular method to detect ermB in clinical isolates to guide therapeutic treatment. Methods: Five sets of loop-mediated isothermal amplification (LAMP) primers were designed to target 6 or 8 distinct sequences on erm B gene by Primer Explorer V4 soft ware. The optimal primers were selected for rapid detection and chosen to determine the specificity and sensitivity of the LAMP reactions for erm B by using DNA extracted from reference strain API10463. Twenty-six enteric pathogenic bacteria strains were selected for specificity assays. Both the real-time monitoring of turbidity and the chromogenic reaction using calcein/Mn2+ complex were included to determine negative and positive results. The results demonstrated that target DNA was amplified and visualized by the 2 detection methods within 60 minutes at an isothermal temperature of 62°C. Besides, erm B gene in Clostridium difficile from 62 clinical isolates were tested by the developed LAMP assay followed by conventional PCR. Simultaneously, the tcdA, tcdB, cdtA, cdtB, and tetM genes were detected by PCR. Results: A total of 26 bacteria strains were negative for erm B, which indicated the high-specificity of the primers. The detection limit of LAMP was 36.1 pg/μl DNA, and its sensitivity was 10-fold greater than that of conventional PCR. The positive rate of erm B obtained from 62 clinical isolates of Clostridium difficile were 98.4% (61/62) by LAMP, which was completely consistent with PCR. Besides, among the 62 isolates analyzed in this study, 60 were toxin A-positive and toxin B-positive (A+B+), 1 was toxin Anegative but toxin B-positive (A−B+), 1 was toxin A, toxin B and binary toxin-positives (A+B+CDT+). Both the A−B+ isolate and 2 A+B+ isolates were negative for tet M, while the rest of isolates were positive (95.2%,59/62). Conclusion: This study is the first report regarding the application of the LAMP assay for detection of erm B gene in Clostridium difficile. The developed LAMP method is exhibited to be a potentially valuable, sensitive, specific, simple, and inexpensive assay for the rapid detection of erm B.

Estimation of teaching-learning-based optimization primer design using regression analysis for different melting temperature calculations.

Primers plays important role in polymerase chain reaction (PCR) experiments, thus it is necessary to select characteristic primers. Unfortunately, manual primer design manners are time-consuming and easy to get human negligence because many PCR constraints must be considered simultaneously. Automatic programs for primer design were developed urgently. In this study, the teaching-learning-based optimization (TLBO), a robust and free of algorithm-specific parameters method, is applied to screen primers conformed primer constraints. The optimal primer frequency (OPF) based on three known melting temperature formulas is estimated by 500 runs for primer design in each different number of generations. We selected optimal primers from fifty random nucleotide sequences of Homo sapiens at NCBI. The results indicate that the SantaLucia's formula is better coupled with the method to get higher optimal primer frequency and shorter CPU-time than the Wallace's formula and the Bolton and McCarthy's formula. Through the regression analysis, we also find the generations are significantly associated with the optimal primer frequency. The results are helpful for developing the novel TLBO-based computational method to design feasible primers.

DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.

The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites.

Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted. By initially defining an objective function, the possible optimal degenerate PCR primer profiles could be automatically explored using the heuristic approach of Greedy Best-First-Search. Compared to the previously developed DC-Analyzer, MDC-Analyzer allows for the existence of a small amount of undesired sequences as a tradeoff between the number of degenerate primers and the encoded library size while still providing all the benefits of DC-Analyzer with the ability to randomize multiple contiguous sites. MDC-Analyzer was validated using a series of randomly generated mutation schemes and experimental case studies on the evolution of halohydrin dehalogenase, which proved that the MDC methodology is more efficient than other methods and is particularly well-suited to exploring the sequence space of proteins using data-driven protein engineering strategies.

Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

Identification of Hucul mare families by mtDNA markers

Hundred animal species have disappeared during the last century. By this time, approximately one-third of domestic animals have been in the endangered category. Hucul horses are also in this category; furthermore saving the genetic diversity beside the race preservation is an important challenge as well. The number of mares and stallions is only one of the expressive elements of genetic diversity; together with their quality determine the genetic variability of this breed. Beyond that, if an exact breed can originates from more founders, it can be more renewed genetically. Stud book documents these data by registering the mare families and stallions’ genealogical lineage. Molecular genetics, especially mitochondrial DNA analysis can make the precise identification of mare families possible. As a result of these molecular based methods, protection of genetic diversity, as well as breed preservation became more reliable. After the primer designing, the optimal primer pair was chosen which targets a 1092 bp length DNA sequence in the cytochrome b region. After the successful PCR optimalisation, we determined 170 Hucul mares’ sequences. According to our results, the samples compose ten haplotypes, which are much less, than the registered number of mare families in the stud book. Further investigations are needed to reach more representative results, and drawn the further consequences.

Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR.

Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R) RT-PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard technique employed by avian immunologists interested in examining these responses. This technique utilizes nucleotide primers and fluorescent reporter molecules to measure amplification of the gene of interest. The use of RRT-PCR negates the need for northern blot analysis or DNA sequencing. It is simple, specific and sensitive for the gene of interest. However, it is dependent on knowing the target sequence prior to testing so that the optimal primers can be designed. The recent publication of genomic sequences of Gallus gallus, Meleagris gallopavo, and Anas platyrhynchos species makes it possible to measure cytokine expression in chicken, turkey, and duck species, respectively. Although these tests do not measure functionally expressed protein, the lack of antibodies to identify and quantify avian cytokines from different avian species makes this technique critical to any characterization of innate immune responses through cytokine and interferon activation or repression.

Microarrays for high‐throughput genotyping ofMICAalleles using allele‐specific primer extension

The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.