转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

5‐Methyl‐2′‐deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson–Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O⁶‐modified 2′‐deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site‐specific 5mC detection, for example, through real‐time PCR approaches.     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

Biofunctionalization of Polyoxometalates with DNA Primers,Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products

The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW₁₁O₃₉{Sn(CH₂)₂CO}]^8? and [P₂W₁₇O₆₁{Sn(CH₂)₂CO}]^6? have been used to link to a 5′‐NH₂ terminated 21‐mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM‐labeled primers was demonstrated through hybridization with a surface‐immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.     

Determination of Yersinia enterocolitica in Food by Capillary Electrophoresis with Laser Induced Fluorescence Detection

A duplex polymerase chain reaction (PCR)-capillary electrophoresis-laser induced fluorescence (CE-LIF) method was developed to determine Yersinia enterocolitica in food sensitively, rapidly, and reliably. Two sets of primers were selected to amplify the genus-specific 16 S ribosomal RNA gene and ail gene associated with the pathogenicity of Yersinia enterocolitica. The parameters of duplex PCR and the conditions for CE-LIF were optimized. Under the optimum conditions, the PCR products of Yersinia enterocolitica were determined within twenty minutes. Alignment analysis showed favorable agreement with published sequences from GenBank, indicating that the primers were specific and the PCR results were reliable. The method detected 16 colony forming units per milliliter pathogenic Yersinia enterocolitica. The intraday precision of migration time of the DNA marker and the PCR products were between 1.13 and 1.81 percent. In summary, a new method combining duplex PCR and CE-LIF is reported for specific, sensitive, and reproducible detection of Yersinia enterocolitica in food with low sample consumption and cost.     

Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN^N:NaO^O), having four hydrogen bonds was investigated. Kinetic studies of single‐nucleotide insertion revealed that ImN^N and NaO^O were recognized as complementary bases by an exonuclease‐deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN^O:NaO^N and ImO^N:NaN^O) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen‐bonding pattern of the ImN^N:NaO^O pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN^N:NaO^O pair was achieved by using DNA polymerases possessing 3′→5′ exonuclease activity (≈99.5 % per doubling).     

High‐Density DNA Functionalization by a Combination of Cu‐Catalyzed and Cu‐Free Click Chemistry

We report the regioselective Cu‐free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3‐Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5‐disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu‐free and Cu‐catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high‐density functionalization of DNA is simple, modular, and efficient.     

Rapid identification of cervus antlers by species-specific PCR assay

Abstract

A rapid PCR technology was developed to differentiate Cervus antlers species and adulteration based on the difference in mitochondrial genome. Three specifically designed primer sets were confirmed to have high inter-species specificity and good intra-species stability. Limits of detection were estimated to be 1?ng of genomes for reindeer and 10?ng for the other species. Especially, when the mixture of Cervus antlers and reindeer or sambar was assayed, these primer sets still exhibited strong capability of differentiation but not the conventional COI barcoding. By using the newly developed approach, five batches out of fourteen commercial Cervus antler products were identified to be fake products made from reindeer antlers. It has shown its good potential to be extensively applied in the identification of counterfeits or adulterates of Cornu Chinese medicines for their pulverized and processed form, and even the traditional Chinese patent medicines composed of these species.