Noninvasive early diagnosis of colorectal cancer is conducive for patients to reduce the mortality rate and their suffering from diagnosis procedures.One of the most significant methods to proceed noninvasive early diagnosis of colorectal cancer is analysis of stool DNA.Extracting high quality and great quantity of human genomic DNA from stools guarantees diagnosis accuracy.However,the complexity of stool component hinders DNA extraction.Hence,it is crucial to develop highly efficient extraction methods of human genomic DNA from stools for the DNA analysis-based early diagnosis of colorectal cancer.Currently,two kinds of extraction strategies are employed:one is to directly extract total DNA from stools,the other is to enrich exfoliated colonocytes in stools before DNA extraction.This article reviews the advances on these two kinds of extraction techniques and summarizes their applications.

The modern large-scale pyrosequencing technology is a revolution of DNA sequencing.One of the key points in this technology is to get an ATP sulfurylase immobilized on the surface of magnetic beads and with a high activity.Biotinylated ATP sulfurylase can be immobilized on magnetic beads coated with streptavidin through the specific conjunction between biotin and streptavidin, but using chemical modification method to biotinylate ATPS will affect the activity of the enzyme.ATP sulfurylase fused with the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein(BCCP87) was expressed in E.coli using fusion expression strategy.Results from Western blot analysis and SDS-PAGE analysis showed that the fusion protein could be biotinylated in vivo, and the molecular mass of the fusion protein was about 64 ku.The biotinylated ATP sulfurylase could be immobilized on the surface of magnetic beads coated with strepavidin, and the immobilized ATPS could be used for quantification of PPi and pyrosequencing.An effective enzyme for the large-scale chip-based pyrosequencing system was supplied.

Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.

Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.
Actinomyces ; enzymology ; Escherichia coli ; genetics ; metabolism ; Pyruvate, Orthophosphate Dikinase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis

Objective The NDRG4 gene methylation in stool is a candidate biomarker for non?invasive diagnosis of colorectal cancer. However, the traditional methods for methylation detection could not be well applied to stool samples due to the low sensitivity and low specificity. The aim of this study was to develop a highly sensitive and specific method for quantifying the methylated NDRG4 gene in stools. Methods Forty one stool samples were collected from 12 colorectal cancer patients, 4 adenoma patients and 25 nor?mal persons. The invasive reaction was combined with real?time PCR and the relative quantification was performed by 2-ΔCT method to develop the highly sensitive and specific methylated DNA detection method, which was used for detecting NDRG4 methylation levels in 41 of stool samples. Results The sensitivity of the method was as low as 10 copies of methylated NDRG4 gene fragments. The specificity was high enough to distinguish 0.01% of methylated fragments from un?methylated fragments and 105 copies of unmethylated NDRG4 fragments gave noamplification signals. The detection results from 41 of stool samples showed that detection rate of the NDRG4 gene in stool from adenoma and colorectal cancer groups had a significant difference comparing to that from the normal group. Conclusion The 2-ΔCT method could accurately quantify the methylation levels of the NDRG4 gene in stool samples, and provide an efficient tool for non?invasive colorectal cancer detection.

A method for the real-time polymerase chain reaction ( PCR ) coupled with high specific invader assay to detect single nucleotide polymorphism ( SNP) was established. To reduce the background signal, the amount of flap endonuclease 1 ( FEN1 enzyme ) and wild-type detection probe was optimized. Under the optimum conditions including 0. 05 μmo/L invasive oligonucleotide probe, 0. 125 μmol/L wild-type detection probe, 0. 5 μmol/L mutation detection probe, 0. 25 μmol/L each fluorescence resonance energy transfer (FRET) probe and 1. 5 U FEN1, the background signal of wild-type sample and mutation sample was dramatically decreased and the background interference to the detecting results was thus eliminated. A total of 21 cases of aldehyde dehydrogenase-2*2 ( ALDH2*2 ) , 19 cases of cytochrome p450 2 C19*2 ( CYP2 C19*2 ) and 19 cases of CYP2C19*3 were analyzed with the established method, and the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote;and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with those by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for the detection of SNP genotyping with non-polluting in single closed tube.

We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
DNA-Binding Proteins ; biosynthesis ; genetics ; Diphosphates ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Animals ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fireflies ; enzymology ; Luciferases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

There are significant individual differences in the antiplatelet effects of aspirin.Three single nucleotide polymorphisms (SNPs),rs5918,rs12041331 and rs730012,are reported to significantly correlate with the efficacy and side effects of aspirin.In the present study,the genotyping method of the three SNPs was established based on the combination of polymerase chain reaction and pyrosequencing technology.Amplification and sequencing primers were designed independently;the amplification conditions were optimized to amplify the three SNPs in the same condition.The sensitivity of the method was detected using original genome DNA at different concentrations.In order to testify the accuracy of the method,the proposed method and Sanger sequencing technology were both used to genotype the three SNPs in 20 blood samples.The results demonstrated that the genotyping method of aspirin-related SNPs was successfully established,with the detection limit being as low as 0.4 ng genome DNA.The genotype results of 20 samples by the proposed method were exactly the same as that of Sanger sequencing.It is evident that the proposed method is sensitive and accurate.

The corona virus disease 2019 (COVID-19) caused by the new coronavirus (SARS-CoV-2) has spread rapidly around the world,posing a serious threat to the public"s health. As of September 30,2020,the number of infected people in the world has reached 33 million,causing more than 1 million deaths. Normalized nucleic acid detection methods based on lab have long turnaround time and high cost. Therefore,there is an urgent need to develop a convenient method to detect SARS-CoV-2,so as to achieve rapid testing and timely control of the epidemic when resources are limited.This review summarizes the point-of-care testing (POCT) methods developed for SARS-CoV-2 in terms of extraction,amplification and detection,and briefly introduces commercial POCT instruments that integrate these three steps,in order to provide references for emergency response and rapid deployment of COVID-19 and other emerging infectious diseases.