Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples.

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Simultaneous quantification of multiple single nucleotide variants in PIK3CA ctDNA using mass-tagged LCR probe sets.

Circulating tumor DNA (ctDNA) in blood carries genetic variations associated with tumors. There is evidence indicating that the abundance of single nucleotide variant (SNV) in ctDNA is correlated well with cancer progression and metastasis. Thus, accurate and quantitative detection of SNVs in ctDNA may benefit clinical practice. However, most current methods are unsuitable for the quantification of SNV in ctDNA that usually differentiates from wild-type DNA (wtDNA) only by a single base. In this setting, ligase chain reaction (LCR) coupled with mass spectrometry (MS) was developed to simultaneously quantify multiple SNVs using PIK3CA ctDNA as a model. Mass-tagged LCR probe set for each SNV including mass-tagged probe and three DNA probes was firstly designed and prepared. Then, LCR was initiated to discriminate SNVs specifically and amplify the signal of SNVs in ctDNA selectively. Afterward, a biotin-streptavidin reaction system was used to separate the amplified products, and photolysis was initiated to release mass tags. Finally, mass tags were monitored and quantified by MS. After optimizing conditions and verifying performance, this quantitative system was applied for blood samples from breast cancer patients, and risk stratification for breast cancer metastasis was also performed. This study is among the first to quantify multiple SNVs in ctDNA in a signal amplification and conversion manner, and also highlights the potential of SNV in ctDNA as a liquid biopsy marker to monitor cancer progression and metastasis.

Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.

Integrated microRNA and transcriptome profiling reveal key miRNA-mRNA interaction pairs associated with seed development in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat seed development is an extremely complex process involving many gene regulatory pathways. MicroRNAs (miRNAs) have been identified as the important negative regulators of gene expression and performed crucial regulatory roles in various plant biological processes. However, whether miRNAs participate in Tartary buckwheat seed development remains unexplored. RESULTS: In this study, we first identified 26 miRNA biosynthesis genes in the Tartary buckwheat genome and described their phylogeny and expression profiling. Then we performed small RNA (sRNA) sequencing for Tartary buckwheat seeds at three developmental stages to identify the miRNAs associated with seed development. In total, 230 miRNAs, including 101 conserved and 129 novel miRNAs, were first identified in Tartary buckwheat, and 3268 target genes were successfully predicted. Among these miRNAs, 76 exhibited differential expression during seed development, and 1534 target genes which correspond to 74 differentially expressed miRNAs (DEMs) were identified. Based on integrated analysis of DEMs and their targets expression, 65 miRNA-mRNA interaction pairs (25 DEMs corresponding to 65 target genes) were identified that exhibited significantly opposite expression during Tartary buckwheat seed development, and 6 of the miRNA-mRNA pairs were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and ligase-mediated rapid amplification of 5' cDNA ends (5'-RLM-RACE). Functional annotation of the 65 target mRNAs showed that 56 miRNA-mRNA interaction pairs major involved in cell differentiation and proliferation, cell elongation, hormones response, organogenesis, embryo and endosperm development, seed size, mineral elements transport, and flavonoid biosynthesis, which indicated that they are the key miRNA-mRNA pairs for Tartary buckwheat seed development. CONCLUSIONS: Our findings provided insights for the first time into miRNA-mediated regulatory pathways in Tartary buckwheat seed development and suggested that miRNAs play important role in Tartary buckwheat seed development. These findings will be help to study the roles and regulatory mechanism of miRNAs in Tartary buckwheat seed development.

Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV.

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction.

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.

Colorimetric detection of class A soybean saponins by coupling DNAzyme with the gap ligase chain reaction.

Class A saponins are responsible for the taste of soybean products, and the rapid identification of class A saponins from soybean food is essential for both food safety and cultivar screening. In this study, we propose a colorimetric assay based on the coupling of gap ligase chain reaction (Gap-LCR) with DNAzyme to detect the target GmSg-1 genes of class A soybean saponins with the naked eye, without the involvement of expensive instruments. The limits of detection (LODs) for the GmSg-1a and GmSg-1b genes were determined to be 0.1618 and 0.1625 µM, respectively, with a linear range of 0.2-1.2 µM. The DNAzyme-based Gap LCR assay was successfully employed to identify the target genes from different soybean cultivars, providing a simple means for monitoring the quality of soybean products.

A novel ligase chain reaction-based electrochemical biosensing strategy for highly sensitive point mutation detection from human whole blood.

Challenged by the detection of trace amounts of mutants and disturbance from endogenous substances in clinical samples, herein, we present a novel electrochemical biosensor based on ligase chain reaction (eLCR) via the thermostable ligase with high mutation recognizing ability. The lengthened double-stranded DNAs exponentially generated via LCR were uniformly distributed on a bovine serum albumin-modified gold electrode, in which the phosphate buffer was tactfully added to remove adsorbed uninterested-probes, and thereafter the amperometry current was collected for the specific binding of streptavidin-poly-HRP and subsequent catalysis in the 3, 3', 5, 5'-tetramethylbenzidine substrate that contained hydrogen peroxide. It found that, under optimized conditions, the proposed biosensor exhibited a high selectivity of mutant targets from the 104-fold excess of co-existent wild targets within a detection limit of 0.5 fM. Impressively, without the involvement of pre-PCR, the homozygous mutants were specifically distinguished from the wild genotype of CYP2C19*2 allele in human whole blood samples. Therefore, the proposed eLCR, due to its advantages in simple primer design, operational ease and ease of miniaturization, has demonstrated its considerable potential for point-of-care testing in the diagnosis of point mutation-related diseases and personalized medicine.

Ferrocene-labeled and purification-free electrochemical biosensor based on ligase chain reaction for ultrasensitive single nucleotide polymorphism detection.

Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A', and B', where B' is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A'B' in 2n. Subsequently, immobilization of the Fc-tagged duplex AB-A'B' on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A'B', such strategy realized directly analysis of LCR products without the need for purification. By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM-10 pM), and the recovery rates (95.3%-107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.

Real-time quantification of fusion transcripts with ligase chain reaction by direct ligation of adjacent DNA probes at fusion junction.

Gene fusions, produced by aberrant juxtapositions of two or more genes even in different chromosomes, play important roles in the primary oncogenic mechanism and have been demonstrated to be typically associated with many cancers. So the fused genes or the transcripts can be specific predictive biomarkers for cancer diagnosis and therapy. Herein, we develop a direct ligation- and ligase chain reaction (LCR)-based method for a fusion transcript assay. In virtue of the high selectivity of ligase and the exponential amplification capacity of LCR, the proposed method can detect as low as 1 fM fusion transcripts with high specificity and has been successfully applied to real samples. With the real-time fluorescence measurements, the fusion transcripts can be assayed in a simple way. Therefore, the proposed method can provide a simple and cost-effective platform for fusion transcript detection in routine laboratories and clinical diagnosis.

"Missing mutations" in MPS I: Identification of two novel copy number variations by an IDUA-specific in house MLPA assay.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is a rare, recessively inherited lysosomal storage disorder, characterized by progressive multi-systemic disease. It is caused by a reduced or absent alpha-l iduronidase (IDUA) enzyme activity secondary to biallelic loss-of-function variants in the IDUA. Over 200 causative variants in IDUA have been identified. Nevertheless, there is a fraction of MPS I patients with only a single mutated IDUA allele detectable. METHODS: As genetic testing of MPS I is usually based on sequencing methods, copy number variations (CNVs) in IDUA can be missed and therefore presumably remain underdiagnosed. The aim of this study was the detection of CNVs using an IDUA-specific in house multiplex ligation-dependent probe amplification (MLPA) assay. RESULTS: A total of five unrelated MPS I patient samples were re-analyzed after only a single heterozygous IDUA mutation c.979G>C (p.A327P), c.1469T>C (p.L490P), c.1598C>G (p.P533R), c.1205G>A (p.W402X), c.973-7C>G (p.?) could be identified. We detected a novel splice site variant c.973-7C>G (p.?), as well as two novel CNVs, a large deletion of IDUA exon 14 and 3'UTR c.(1828 + 1_1829-1)_(*1963_?)del, and a large duplication extending from IDUA exon 2 to intron 12 c.(157 + 1_158-1)_(1727 + 1_1728-1)dup. CONCLUSION: Together with the CNVs we previously identified, a total of four pathogenic IDUA CNVs have now been reported.

Frequency and characterization of RHD variants in serologically D- Surinamese pregnant women and D- newborns.

BACKGROUND: Numerous RHD variant genes affect the expression of D on the red blood cell surface. In Suriname, 4.3% of pregnant women were D-, ranging from virtually zero to 7% among ethnic groups. Characterization of RHD variants, which are associated with a variable potential to induce anti-D, is of practical clinical importance especially in case of limited access to preventive measures. Here we report on the occurrence of RHD variant genes in Surinamese serologically D- pregnant women and their D- newborns from different ethnic groups. STUDY DESIGN AND METHODS: The RheSuN study is a cross-sectional cohort study in D- pregnant women and their newborns, who visited hospitals in Paramaribo, Suriname, during routine pregnancy care. The presence of RHD variants was investigated using quantitative polymerase chain reaction targeting RHD Exons 5 and 7 and RH-multiplex ligation-dependent probe amplification. RESULTS: Seven RHD variant genes were detected in 35 of 84 women and four RHD variant genes in 15 of 36 newborns. The RHD*03 N.01 and RHD*08 N.01 variants represented 87% of a total of 62 variant genes. Variants were comparably frequent among ethnicities. In four cases genotyping would have changed anti-D prophylaxis policy: one woman with a RHD*01EL.01 variant, not associated with anti-D formation and three D- newborns with RHD*09.01 and RHD*09.03.01 variants, potentially capable of inducing anti-D. CONCLUSION: RHD variants at risk for anti-D are common among serologic D- individuals from African descent in Suriname. While genotyping D- women has limited added value, it may be considered in newborns from D- women.

Homogentisate 1,2-dioxygenase (HGD) gene variants, their analysis and genotype-phenotype correlations in the largest cohort of patients with AKU.

Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligation-dependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotype-phenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.

Development of a Polymerase Chain Reaction/Ligase Detection Reaction Assay for Detection of CYP2C19 Polymorphisms.

AIMS: Cytochrome P450 2C19 (CYP2C19) genotypes are associated with differential drug metabolism. The aim of this study was to establish a reliable assay for CYP2C19 genotyping based on a polymerase chain reaction/ligase detection reaction (PCR-LDR). MATERIALS AND METHODS: Specific primers and probes were designed to detect CYP2C19*1, *2, *3, and *17. A control for each allele was prepared and used for performance evaluation. A total of 200 clinical samples were analyzed using the PCR-LDR assay and Sanger sequencing. RESULTS: The detection limit of the PCR-LDR assay was 2 ng/µL of genomic DNA. Common interfering substances in the blood did not affect the results of the detection. For the clinical samples, the results of the PCR-LDR and the Sanger sequencing were identical. Among the 200 patients, 104 (52%) were wild type (*1/*1), 64 (32%) were *1/*2, 16 (8%) were *1/*3, 8 (4%) were *2/*2, 7 (3.5%) were *2/*3, and 1 (0.5%) was *1/*7. No *3/*3 genotype was detected in these patients. CONCLUSION: This PCR-LDR assay is reliable for the detection of CYP2C19 genotypes in a clinical setting. It will be a useful tool to screen for CYP2C19 loss-of-function alleles in patients before clopidogrel and proton pump inhibitor treatment.

Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications.

Genetic variants have been reported to cause several genetic diseases. Various genotyping assays have been developed for diagnostic and screening purposes but with certain limitations in sensitivity, specificity, cost effectiveness and/or time savings. Since the discovery of ligase chain reaction (LCR) in the late nineties, it became one of the most favored platforms for detecting these variants and also for genotyping low abundant contaminants. Recent and powerful modifications with the integration of various detection strategies such as electrochemical and magnetic biosensors, nanoparticles (NPs), quantum dots, quartz crystal and leaky surface acoustic surface biosensors, DNAzyme, rolling circle amplification (RCA), strand displacement amplification (SDA), surface enhanced raman scattering (SERS), chemiluminescence and fluorescence resonance energy transfer have been introduced to both LCR and ligation based amplifications to enable high-throughput and inexpensive multiplex genotyping with improved robustness, simplicity, sensitivity and specificity. In this article, classical and up to date modifications in LCR and ligation based amplifications are critically evaluated and compared with emphasis on points of strength and weakness, sensitivity, cost, running time, equipment needed, applications and multiplexing potential. Versatile genotyping applications such as genetic diseases detection, bacterial and viral pathogens detection are also detailed. Ligation based gold NPs biosensor, ligation based RCA and ligation mediated SDA assays enhanced detection limit tremendously with a discrimination power approaching 1.5aM, 2aM and 0.1fM respectively. MLPA (multiplexed ligation dependent probe amplification) and SNPlex assays have been commercialized for multiplex detection of at least 48 SNPs at a time. MOL-PCR (multiplex oligonucleotide ligation) has high-throughput capability with multiplex detection of 50 SNPs/well in a 96 well plate. Ligase detection reaction (LDR) is one of the most widely used LCR versions that have been successfully integrated with several detection strategies with improved sensitivity down to 0.4fM.

Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction.

The 5'-cap structure is an essential feature in eukaryotic mRNA required for mRNA stability and enhancement of translation. Ceratin transcripts are selectively silenced by decapping in the cytoplasm and later become translationally active again by acquiring the cap structure to regenerate translatable mRNAs. Identification of uncapped mRNA transcripts will reveal how gene expression is regulated by the mRNA recapping pathway. What follows is a sensitive method to detect and identify the uncapped mRNA from the cells. The technique consists of three parts: selective ligation of anchor RNA to the 5'-end of monophosphate RNA by double-strand RNA ligase, conversion of ligated RNA product into cDNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction.

Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout-ExoPLA.

Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc.

Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis.