Structural bias in T4 RNA ligase-mediated 3'-adapter ligation

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3'-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3'-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3'-adapter ligation step.     

TE-AFLP: combining rapidity and robustness in DNA fingerprinting

A new type of fingerprinting technique is presented, based on amplified fragment length polymorphism (AFLP). Rather than two endonucleases as in AFLP, we propose the use of three enzymes, hence the method is called three endonuclease (TE)-AFLP. Genomic DNA is digested and two sets of adapters are selectively ligated onto the restriction fragments in a single reaction volume. No adapters complementary to the ends generated by a frequent cutter are added. Due to the addition of a third endonuclease, the TE-AFLP method provides a high discriminatory power and a reduction in the number of bands. The latter makes it especially suitable for the analysis of complex genomes. TE-AFLP fingerprints are suitable for detection by automatic fluorescent sequencers and are obtained in less than half the time and at reduced costs compared to a typical AFLP. The reliability of this method was investigated by determining the influence of varying digestion, ligation and PCR components on the fingerprint. Moreover, cross-experiments to study inheritance of loci were performed with a primitive insect and with tomato strains. The features of TE-AFLP are discussed in comparison with conventional AFLP.     

Selective DNA amplification from complex genomes using universal double-sided adapters

There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest.     

AFLP分子标记技术及其在动物学研究中的应用

扩增片段长度多态性技术(AFLP)基于选择性扩增完全酶切消化后的基因组DNA片段,包括酶切与连接、选择性扩增、检测分析等3个步骤。该技术的运用不需要预知基因组的序列特征,具有较高的多态分辨力,产生的标记是显性标记,可适用于任何来源和各种复杂度的DNA。自AFLP技术问世以来,在酶切、扩增体系、检测和分析方法等方面不断得到改进。本文将以线虫、昆虫、鱼类、鸟类、家畜、鼠、人等为例,介绍近年来AHLP技术在动物或人的遗传图谱构建和QTL(quantitative trait loci)定位、生物多样性、性别决定和繁殖行为研究、疾病及疾病诊断研究等上的应用。     

蛋白质多肽文库构建中限制性显示技术的接头设计

为了探讨限制性显示(RD)技术在构建蛋白质多肽文库中灵活的接头设计,分别根据原核表达载体pET22b以及酵母表达载体pNMT-TOPO设计了三套接头,三套接头依次增加一个碱基以保证与之连接的片段总有可能表达正确的开放阅读框.然后以HIV-1 B亚型代表株U26942全基因质粒DNA为对象,利用RD技术分别建立了相应的蛋白质多肽文库.从每个库中各随机挑选12个克隆进行测序分析并进行蛋白质表达预测.结果从原核表达文库中获得了一个可以表达HIV Pol多肽的克隆,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示该克隆在细菌BL21(DE3)中有较高的表达,蛋白质印迹为阳性,与理论预测相符.这些结果提示,RD技术是一种建立基因组随机多肽文库的新方法,该方法灵活的接头设计可以满足不同的表达载体需求.     

Improved method for detecting differentially expressed genes using cDNA indexing

In cDNA indexing, differentially expressed genes are identified by the display of specific, corresponding subsets of cDNA. Subdivision of the cDNA population is achieved by the sequence-specific ligation of adapters to the overhangs created by class IIS restriction enzymes. However, inadequate specificity of ligation leads to redundancy between different adapter subsets. We evaluate the incidence of mismatches between adapters and class IIS restriction fragments during ligation and describe a modified set of conditions that improves ligation specificity. The improved protocol reduces redundancy between amplified cDNA subsets, which leads to a lower number of bands per lane of the differential display gel, and therefore simplifies analysis. We confirm the validity of this revised protocol by identifying five differentially expressed genes in mouse duodenum and ileum.     

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I

Many high-throughput small RNA next-generation sequencing protocols use 5′ preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter''s 5′ end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5′ adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3′ blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3′ ends to be 5′ adenylylated at >90% efficiency.     

Formation of MboII vectors and cassettes using asymmetric MboII linkers

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.     

应用RD-PCR技术制备基因芯片靶基因片段

应用RD－PCR技术分离SH－SY5Y细胞的基因片段。从正常培养的SH－SY5Y细胞中提取总RNA,经oligo(dT）纤维素柱纯化分离出mRNA,然后以oligo(dT18)为锚定引物反转录生成单链cDNA,再以此为模板合成DNA的第二条链;将双链DNA经Sau3AI酶切之后,接上接头,经通用引物和选择性引物进行扩增;然后与载体pMD18-T相连,克隆鉴定、筛选、测序。所分离的cDNA片段经过扩增后用于制备基因芯片的靶基因,杂交检测的结果表明,此种方法所分离的基因片段可以用于基因芯片的靶基因片段,所制备的芯片将为进一步研究神经细胞基因表达提供了条件。     

A simple, effective method for the construction of subtracted cDNA libraries

A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.     

A PCR-based DNA fingerprinting technique: AFLP for molecular typing of bacteria.

Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique. In AFLP analysis, bacterial genomic DNA is digested with restriction enzymes, ligated to adapters, and a subset of DNA fragments are amplified using primers containing 16 adapter defined sequences with one additional arbitrary nucleotide. Polymorphisms of different Escherichia coli strains or Agrobacterium tumefaciens strains were demonstrated as distinct, unique bands in a denaturing sequencing gel using AFLP. The polymorphisms detected between BL21 and BL21F'IQ and between DH5 alpha and DH5 alpha F'IQ strains indicated that AFLP is able to resolve differences in F' episomal DNA (approximately 100 kb).     

Universal restriction site-free cloning method using chimeric primers

A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the DNA fragment or the vector. The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector. The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La3+ or Lu3+. Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3'overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3' overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.     

AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.     

Studies on Microdissection and Microcloning of the Rye Chromosome 1R

Two 1R chromosomes of Secale cereale L. were isolated from one metaphase cell by means of chromosome micro-isolation, and the chromosomal DNA was amplified adopting the cohesive adapters single primer polymerase chain reaction (CASP-PCR) technique. The CASP-PCR products were labeled as probes. The results of Southern blot hybridization confirmed that the CASP- PCR products derived from the chromosome IR were homologous with the genomic DNA of S. cereale. The clones of PCR products were obtained with high efficiency. Over 10 000 recombinant clones were obtained from one-tenth of the ligation mixture which was transferred into the competent E. coli DH5a. The size of the inserted fragments of clones ranged from 250 bp to 500 bp. This research has established the foundation for further selection of chromosome 1R markers.     

应用PCR-酶切连接法合成全长sFat1基因

人工合成基因在生命科学研究中有着重要的意义, 因此基因合成是一项常用技术。长片段基因的合成比较困难, 常常因为合成中碱基序列的错配、突变等原因而导致失败。研究者们所熟知的几种现行的方法仍然难以解决该问题。本研究在作者自身的工作经验中建立了一种新的基因合成方法, 即PCR-酶切连接法。应用该方法成功地将化学合成的27个寡聚核苷酸片段(每个片段长60～68 bp)拼接组装起来, 获得了完整的总长为1 226 bp的基因sFat-1。整个过程仅采用3轮PCR(共7个反应)、2轮的酶切连接(3个反应), 而且未曾出现任何偏离预期基因序列的差错。该方法步骤较少, 技术简单, 出错极少, 是合成长基因序列很好的选择。     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998     

A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.