The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in ...The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.展开更多
The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended b...The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.展开更多
Based on the fact that the resonant frequency of a piezoelectric crystal is the function of its surface deposit, and that the primer extends after it hybridizes with the template, the primer extension gene sensor tech...Based on the fact that the resonant frequency of a piezoelectric crystal is the function of its surface deposit, and that the primer extends after it hybridizes with the template, the primer extension gene sensor technique was developed. The prominent feature of the technique is that fast and sensitive frequency signals are used as the monitoring system of gene hybridization and primer strand extension. Results show that this technique may be used in homologous analysis of nucleic acid, trace DNA detection, and determining the integration of DNA. It may also be used for isolation of target gene, gene mutation analysis, and predicting the location of a gene in its genome.展开更多
Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions.Primer extension reactions are commonly used for the assay of such reaction events.However,current assay protocols ge...Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions.Primer extension reactions are commonly used for the assay of such reaction events.However,current assay protocols generally rely on radiolabeling,fluorescence reporter labeling,or removal of specific deoxynucleotide triphosphate in the reaction mixture.Herein we report on the design of two novel assay protocols that utilize a dideoxynucleo-tide-terminated template strand and a phosphorothiolate-modified deoxynucleotide-terminated template strand.We designed and synthesized a deoxyuridine triphosphate analogue(dU^(*)TP)containing 2-bromoisobutyryl group and demonstrated that it could be well recognized byφ29DNA polymerase,E.coli DNA polymerase I Klenow Fragment,Bst DNA polymerase Large Fragment,and E.coli DNA polymerase I Klenow Fragment(exo^(−)),which translated to effective incorporation of dU^(*)TP into DNA.dU^(*)TP was also successfully incorporated into extremely long single-stranded DNA at high-density usingφ29 DNA polymerase by rolling circle amplification.展开更多
生物芯片技术是生物技术和微制造技术的融合, 已广泛用于生命科学的研究及实践、医学科研及临床、药物设计、环境保护、农业、军事等各个领域。而基因芯片是生物芯片中的一种,是指将大量基因探针分子固定于支持物上,然后与标记的样品进...生物芯片技术是生物技术和微制造技术的融合, 已广泛用于生命科学的研究及实践、医学科研及临床、药物设计、环境保护、农业、军事等各个领域。而基因芯片是生物芯片中的一种,是指将大量基因探针分子固定于支持物上,然后与标记的样品进行杂交,所以一次可对大量核酸分子进行检测分析,从而解决了传统核酸印迹杂交技术操作复杂、自动化程度低、检测目的分子数量少、效率低等不足。文章探讨了用基因芯片这一新的检测手段对转基因植物的初步检测,采用一种新的反应机制-引物延伸芯片法(arrayed primer extension),实现了样品扩增和杂交的一步化,而在传统的基因芯片检测中要需要两步来完成,从而为目前基因芯片中大片段样品的检测提供了一种可能性。展开更多
采用RNA加尾和引物延伸real time PCR法实时定量检测了黑腹果蝇Drosophila melanogaster的bantam、mir-14、mir-2a共3种microRNA（miRNA）在各发育阶段组织中的相对表达情况,发现3种miRNA在不同发育时期的表达差异明显,其表达变化规律...采用RNA加尾和引物延伸real time PCR法实时定量检测了黑腹果蝇Drosophila melanogaster的bantam、mir-14、mir-2a共3种microRNA（miRNA）在各发育阶段组织中的相对表达情况,发现3种miRNA在不同发育时期的表达差异明显,其表达变化规律与文献报道基本一致,验证了该法的可靠性.这种＂加尾和引物延伸＂方法,仅需1~10 ng总RNA或等同物,检测范围可达7个数量级,为昆虫miRNA研究提供了可靠的定量检测方法.展开更多
基金Financial support received from Department of Biotechnology,Government of India vide grant No.BT/PR-8953/BCE/08/533/2007project sanctioned against grant No.BT/04/NE/2009financial support from Department of Science&Technology,Government of India in the form of a research fellowship under the INSPIRE program
文摘The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.
基金the National Natural Sci-ence Foundation of China (No. 60121101)the Hi-Tech Research and Development Program of China (No. 2006AA020702).
文摘The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.
基金Project supported by the National Natural Science Foundation of China.
文摘Based on the fact that the resonant frequency of a piezoelectric crystal is the function of its surface deposit, and that the primer extends after it hybridizes with the template, the primer extension gene sensor technique was developed. The prominent feature of the technique is that fast and sensitive frequency signals are used as the monitoring system of gene hybridization and primer strand extension. Results show that this technique may be used in homologous analysis of nucleic acid, trace DNA detection, and determining the integration of DNA. It may also be used for isolation of target gene, gene mutation analysis, and predicting the location of a gene in its genome.
文摘Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions.Primer extension reactions are commonly used for the assay of such reaction events.However,current assay protocols generally rely on radiolabeling,fluorescence reporter labeling,or removal of specific deoxynucleotide triphosphate in the reaction mixture.Herein we report on the design of two novel assay protocols that utilize a dideoxynucleo-tide-terminated template strand and a phosphorothiolate-modified deoxynucleotide-terminated template strand.We designed and synthesized a deoxyuridine triphosphate analogue(dU^(*)TP)containing 2-bromoisobutyryl group and demonstrated that it could be well recognized byφ29DNA polymerase,E.coli DNA polymerase I Klenow Fragment,Bst DNA polymerase Large Fragment,and E.coli DNA polymerase I Klenow Fragment(exo^(−)),which translated to effective incorporation of dU^(*)TP into DNA.dU^(*)TP was also successfully incorporated into extremely long single-stranded DNA at high-density usingφ29 DNA polymerase by rolling circle amplification.
文摘生物芯片技术是生物技术和微制造技术的融合, 已广泛用于生命科学的研究及实践、医学科研及临床、药物设计、环境保护、农业、军事等各个领域。而基因芯片是生物芯片中的一种,是指将大量基因探针分子固定于支持物上,然后与标记的样品进行杂交,所以一次可对大量核酸分子进行检测分析,从而解决了传统核酸印迹杂交技术操作复杂、自动化程度低、检测目的分子数量少、效率低等不足。文章探讨了用基因芯片这一新的检测手段对转基因植物的初步检测,采用一种新的反应机制-引物延伸芯片法(arrayed primer extension),实现了样品扩增和杂交的一步化,而在传统的基因芯片检测中要需要两步来完成,从而为目前基因芯片中大片段样品的检测提供了一种可能性。
文摘采用RNA加尾和引物延伸real time PCR法实时定量检测了黑腹果蝇Drosophila melanogaster的bantam、mir-14、mir-2a共3种microRNA（miRNA）在各发育阶段组织中的相对表达情况,发现3种miRNA在不同发育时期的表达差异明显,其表达变化规律与文献报道基本一致,验证了该法的可靠性.这种＂加尾和引物延伸＂方法,仅需1~10 ng总RNA或等同物,检测范围可达7个数量级,为昆虫miRNA研究提供了可靠的定量检测方法.
基金supported by 11th 5-Year Plan(2007BAI18B13)the National Natural Science Foundation of China(30671150+1 种基金81170923)“863”Program(2007AA02Z445),P.R.China
