Chunming Ding

Engineered riboregulators enable post-transcriptional control of gene expression

Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5′–untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS

We report here an approach for gene expression analysis by combining competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. A DNA standard is designed with an artificial single nucleotide polymorphism in the gene of interest. The standard is added to the reverse transcription product before PCR. Subsequently, a base extension reaction is carried out at the single nucleotide polymorphism position, and the products are quantified by matrix-assisted laser desorption ionization time-of-flight MS. The approach is capable of relative and absolute quantification of gene expression; it is extremely sensitive (as few as five copies of DNA were quantified) and highly reproducible. It is also capable of simultaneous quantification of both alleles for heterozygotes and alternatively spliced genes. We have incorporated this technique with the homogeneous Mass Extension system (Sequenom) to create a high-throughput, automated gene expression analysis platform where a few hundred genes from 20–500 different samples can be accurately quantified per day.

Systematic search for placental DNA-methylation markers on chromosome 21: Toward a maternal plasma-based epigenetic test for fetal trisomy 21

BACKGROUND The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18. METHODS To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)-76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria-for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site. RESULTS We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22-0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00-0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance. CONCLUSION Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

Noninvasive Prenatal Detection of Trisomy 21 by an Epigenetic–Genetic Chromosome-Dosage Approach

BACKGROUND The use of fetal DNA in maternal plasma for noninvasive prenatal diagnosis of trisomy 21 (T21) is an actively researched area. We propose a novel method of T21 detection that combines fetal-specific epigenetic and genetic markers. METHODS We used combined bisulfite restriction analysis to search for fetal DNA markers on chromosome 21 that were differentially methylated in the placenta and maternal blood cells and confirmed any target locus with bisulfite sequencing. We then used methylation-sensitive restriction endonuclease digestion followed by microfluidics digital PCR analysis to investigate the identified marker. Chromosome-dosage analysis was performed by comparing the dosage of this epigenetic marker with that of the ZFY (zinc finger protein, Y-linked) gene on chromosome Y. RESULTS The putative promoter of the HLCS (holocarboxylase synthetase) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. A chromosome-dosage comparison of the hypermethylated HLCS and ZFY loci could distinguish samples of T21 and euploid placental DNA. Twenty-four maternal plasma samples from euploid pregnancies and 5 maternal plasma samples from T21 pregnancies were analyzed. All but 1 of the euploid samples were correctly classified. CONCLUSIONS The epigenetic-genetic chromosome-dosage approach is a new method for noninvasive prenatal detection of T21. The epigenetic part of the analysis can be applied to all pregnancies. Because the genetic part of the analysis uses paternally inherited, fetal-specific genetic markers that are abundant in the genome, broad population coverage should be readily achievable. This approach has the potential to become a generally usable technique for noninvasive prenatal diagnosis.

Bacterial microbiota profiling in gastritis without Helicobacter pylori infection or non-steroidal anti-inflammatory drug use.

Recent 16S ribosomal RNA gene (rRNA) molecular profiling of the stomach mucosa revealed a surprising complexity of microbiota. Helicobacter pylori infection and non-steroidal anti-inflammatory drug (NSAID) use are two main contributors to gastritis and peptic ulcer. However, little is known about the association between other members of the stomach microbiota and gastric diseases. In this study, cloning and sequencing of the 16S rRNA was used to profile the stomach microbiota from normal and gastritis patients. One hundred and thirty three phylotypes from eight bacterial phyla were identified. The stomach microbiota was found to be closely adhered to the mucosa. Eleven Streptococcus phylotypes were successfully cultivated from the biopsies. One to two genera represented a majority of clones within any of the identified phyla. We further developed two real-time quantitative PCR assays to quantify the relative abundance of the Firmicutes phylum and the Streptococcus genus. Significantly higher abundance of the Firmicutes phylum and the Streptococcus genus within the Firmicutes phylum was observed in patients with antral gastritis, compared with normal controls. This study suggests that the genus taxon level can largely represent much higher taxa such as the phylum. The clinical relevance and the mechanism underlying the altered microbiota composition in gastritis require further functional studies.

Quantitative analysis of circulating methylated DNA as a biomarker for hepatocellular carcinoma

BACKGROUND Hypermethylation of the RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] gene is frequently observed in hepatocellular carcinoma (HCC). We evaluated the analysis of circulating hypermethylated RASSF1A for detecting HCC and assessing its prognosis. METHODS In module 1, we studied 63 pairs of HCC patients and age- and sex-matched chronic hepatitis B virus (HBV) carriers, as well as 50 healthy volunteers. In module 2, we studied 22 HCC patients with cancer detected through a surveillance program. The concentrations of circulating hypermethylated RASSF1A sequences were measured by real-time PCR after digestion with a methylation-sensitive restriction enzyme. RESULTS We detected hypermethylated RASSF1A sequences in the sera of 93% of HCC patients, 58% of HBV carriers, and 8% of the healthy volunteers. The median RASSF1A concentrations for the HCC patients and HBV carriers were 7.70 x 10(5) copies/L and 1.18 x 10(5) copies/L, respectively (P < 0.01). The detection of low concentrations in HBV carriers is consistent with previous findings that RASSF1A hypermethylation is an early event in HCC pathogenesis and can be found in premalignant liver tissues. Use of a marker cutoff value of 1 x 10(6) copies/L also identifies 50% of alpha-fetoprotein-negative HCC cases. Patients with higher RASSF1A concentrations at diagnosis or 1 year after tumor resection showed poorer disease-free survival (P < 0.01). For the HBV carriers who underwent HCC surveillance and subsequently developed HCC, the circulating concentration of RASSF1A increased significantly from the time of enrollment to cancer diagnosis (P = 0.014). CONCLUSIONS Detection and quantification of circulating methylated RASSF1A sequences are useful for HCC screening, detection, and prognostication.

MuPlex: multi-objective multiplex PCR assay design

We have developed a web-enabled system called MuPlex that aids researchers in the design of multiplex PCR assays. Multiplex PCR is a key technology for an endless list of applications, including detecting infectious microorganisms, whole-genome sequencing and closure, forensic analysis and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays is computationally challenging because it involves tradeoffs among competing objectives, and extensive computational analysis is required in order to screen out primer-pair cross interactions. With MuPlex, users specify a set of DNA sequences along with primer selection criteria, interaction parameters and the target multiplexing level. MuPlex designs a set of multiplex PCR assays designed to cover as many of the input sequences as possible. MuPlex provides multiple solution alternatives that reveal tradeoffs among competing objectives. MuPlex is uniquely designed for large-scale multiplex PCR assay design in an automated high-throughput environment, where high coverage of potentially thousands of single nucleotide polymorphisms is required. The server is available at .

Simultaneous Quantitative and Allele-Specific Expression Analysis with Real Competitive PCR

BackgroundFor a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis.ResultsA transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies.ConclusionsThis technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.

Multiplex Detection of 60 Hepatitis B Virus Variants by MALDI-TOF Mass Spectrometry

BACKGROUND Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy. METHODS A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results. RESULTS The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls. CONCLUSIONS This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.