The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modul...The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.展开更多
Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly...Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly chosen individuals, and evenamong siblings. A mtDNA pseudogene in the Ychromosome was observed in a YAC clone carrying onlyrepetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showedthat there were at least 5.7×105 hp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part ofthe human nuclear genome is discussed.展开更多
Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy samp...Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,展开更多
Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited dis...Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.展开更多
Objective: Sequencing of mouse Tbx2 gene andobserving the expression of Tbx2 gene in normal andmalignant melanophore. Methods: The PCR productsof TbX2 cDNA were cloned into PUC18 vector andsequenced. The normal and ma...Objective: Sequencing of mouse Tbx2 gene andobserving the expression of Tbx2 gene in normal andmalignant melanophore. Methods: The PCR productsof TbX2 cDNA were cloned into PUC18 vector andsequenced. The normal and malignant melanocytes wereused to extract total RNA. The expression of Tbx2 genewas detected by RT-PCR. Results: The TbXZ genome iscomposed of seven e-cons and six nitrons. No expressionof Tbx2 gene in the normal melanocytes was noted, butall malignant melanocytes showed expression of TbXZgene. Conclusion: The observation showed the analysisof the genomic structure of mouse TbX2. TbX2 plays acritical role during the development of the malignantmelanophore.展开更多
Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation ...Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation genetic diagnosis(PGD) examine distinct parts of an individua genome, require the development of a custom assay for every patient family, and are time consuming and inefficient. In the last decade technologies for wholegenome amplification(WGA) from single cells have led to innovative strategies for preimplantation testing.Applications of WGA technology can lead to a universa approach that uses single-nucleotide polymorphisms(SNPs) and mutations across the entire genome for the analysis. Single-cell WGA by multiple displacement amplification has enabled a linkage approach to PGD known as "preimplantation genetic haplotyping", as well as microarray-based techniques for preimplantation diagnosis. The use of microarrays in preimplantation diagnosis has provided genome-wide testing for gains or losses of single chromosomes(aneuploidies)or chromosomal segments. Properly designed randomized controlled trials are, however, needed to determine whether these new technologies improve IVF outcomes by increasing implantation rates and decreasing mis-carriage rates. In genotype analysis of single cells, allele dropout occurs frequently at heterozygous loci. Preimplantation testing of multiple cells biopsied from blastocysts, however, can reduce allele dropout rates and increase the accuracy of genotyping, but it allows less time for PGD. Future development of fast SNP microarrays will enable a universal preimplantation testing for aneuploidies, single-gene disorders and unbalanced translocations within the time frame of an IVF cycle.展开更多
Background:The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells,so whole-genome amplification(WGA)is necessary to increase the DNA amount and enable downstream analy...Background:The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells,so whole-genome amplification(WGA)is necessary to increase the DNA amount and enable downstream analyses.Multiple displacement amplification(MDA)is the most widely used WGA technique.Results:Compared with amplification methods based on PCR and other methods,MDA renders high-quality DNA products and better genome coverage by using phi29 DNA polymerase.Moreover,recently developed advanced MDA technologies such as microreactor MDA,emulsion MDA,and micro-channel MDA have improved amplification uniformity.Additionally,the development of other novel methods such as TruePrime WGA allows for amplification without primers.Conclusion:Here,we reviewed a selection of recently developed MDA methods,their advantages over other WGA methods,and improved MDA-based technologies,followed by a discussion of future perspectives.With the continuous development of MDA and the successive update of detection technologies,MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.展开更多
针对烟草属特异性基因Ntsp151序列保守区设计环介导等温扩增(LAMP)引物,基于SYBR Green I荧光染料对烟草材料进行可视化LAMP检测,对检测过程中的DNA提取方法和反应条件进行优化,并对LAMP反应体系的特异性和灵敏度进行评价。结果表明:使...针对烟草属特异性基因Ntsp151序列保守区设计环介导等温扩增(LAMP)引物,基于SYBR Green I荧光染料对烟草材料进行可视化LAMP检测,对检测过程中的DNA提取方法和反应条件进行优化,并对LAMP反应体系的特异性和灵敏度进行评价。结果表明:使用Chelex-100提取的DNA可以直接进行LAMP扩增反应,显色效果较好;LAMP最适反应条件为扩增温度63℃、反应时间60 min、Mg^(2+)浓度6 mmol/L;LAMP对17份非烟草材料和1份烟草材料基因组DNA的扩增显色结果与荧光值的检测结果一致,具有较好的特异性,其最低检测限为10^(2) copies/μL。基于烟草属特异性基因Ntsp151的LAMP检测结果可视化强,且灵敏度高、特异性强,适用于现场抽检。展开更多
Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the featu...Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification （MLPA） and array comparative genomic hybridization （array CGH）. Methods： Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results： All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions： The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.展开更多
文摘The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.
文摘Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly chosen individuals, and evenamong siblings. A mtDNA pseudogene in the Ychromosome was observed in a YAC clone carrying onlyrepetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showedthat there were at least 5.7×105 hp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part ofthe human nuclear genome is discussed.
基金Project(50621063)supported by the National Natural Science Foundation of ChinaProject(2004CB619201)supported by the National Basic Research Program of China
基金supported by grants awarded to Yuanqing Yao by the Key Program of the "Twelfth Five-year plan" of People’s liberation Army(No.BWS11J058)the National High Technology Research and Development Program(SS2015AA020402)
文摘Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,
基金Project supported by the National Basic Research Program (973) of China (No.2007CB948104)the Natural Science Foundation of Zhejiang Province,China (No.Z207021)
文摘Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.
文摘Objective: Sequencing of mouse Tbx2 gene andobserving the expression of Tbx2 gene in normal andmalignant melanophore. Methods: The PCR productsof TbX2 cDNA were cloned into PUC18 vector andsequenced. The normal and malignant melanocytes wereused to extract total RNA. The expression of Tbx2 genewas detected by RT-PCR. Results: The TbXZ genome iscomposed of seven e-cons and six nitrons. No expressionof Tbx2 gene in the normal melanocytes was noted, butall malignant melanocytes showed expression of TbXZgene. Conclusion: The observation showed the analysisof the genomic structure of mouse TbX2. TbX2 plays acritical role during the development of the malignantmelanophore.
基金Supported by Department of Pediatrics,Medical College of Wisconsin,Milwaukee,WI,United States
文摘Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation genetic diagnosis(PGD) examine distinct parts of an individua genome, require the development of a custom assay for every patient family, and are time consuming and inefficient. In the last decade technologies for wholegenome amplification(WGA) from single cells have led to innovative strategies for preimplantation testing.Applications of WGA technology can lead to a universa approach that uses single-nucleotide polymorphisms(SNPs) and mutations across the entire genome for the analysis. Single-cell WGA by multiple displacement amplification has enabled a linkage approach to PGD known as "preimplantation genetic haplotyping", as well as microarray-based techniques for preimplantation diagnosis. The use of microarrays in preimplantation diagnosis has provided genome-wide testing for gains or losses of single chromosomes(aneuploidies)or chromosomal segments. Properly designed randomized controlled trials are, however, needed to determine whether these new technologies improve IVF outcomes by increasing implantation rates and decreasing mis-carriage rates. In genotype analysis of single cells, allele dropout occurs frequently at heterozygous loci. Preimplantation testing of multiple cells biopsied from blastocysts, however, can reduce allele dropout rates and increase the accuracy of genotyping, but it allows less time for PGD. Future development of fast SNP microarrays will enable a universal preimplantation testing for aneuploidies, single-gene disorders and unbalanced translocations within the time frame of an IVF cycle.
基金project 61971125 of the National Natural Science Foundation of China and the Fundamental Research Funds for the Central Universities of China.
文摘Background:The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells,so whole-genome amplification(WGA)is necessary to increase the DNA amount and enable downstream analyses.Multiple displacement amplification(MDA)is the most widely used WGA technique.Results:Compared with amplification methods based on PCR and other methods,MDA renders high-quality DNA products and better genome coverage by using phi29 DNA polymerase.Moreover,recently developed advanced MDA technologies such as microreactor MDA,emulsion MDA,and micro-channel MDA have improved amplification uniformity.Additionally,the development of other novel methods such as TruePrime WGA allows for amplification without primers.Conclusion:Here,we reviewed a selection of recently developed MDA methods,their advantages over other WGA methods,and improved MDA-based technologies,followed by a discussion of future perspectives.With the continuous development of MDA and the successive update of detection technologies,MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.
文摘Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification （MLPA） and array comparative genomic hybridization （array CGH）. Methods： Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results： All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions： The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.
