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URA5-RFLP is one of the most widely used genotyping methods relating to Cryptococcus neoformans and C. gattii consensus genotype nomenclature. In order to identify a molecular type, this method uses a visual comparison of digested PCR products of tested and reference strains, therefore any anomaly in RFLP patterns of studied isolates makes recognition difficult or impossible. This report describes a strain of VNIV type showing an atypical URA5-RFLP pattern as well as a group of AD hybrids displaying the same anomaly. The atypical RFLP pattern is the result of a point mutation and emergence of a new restriction site. Emergence of the allele presenting a new banding pattern may lead to misidentification using the URA5-RFLP technique; the results of this study as well as the literature data may suggest the spread of the allele in the environment.

Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges’ λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based “All-In-One” genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a “LHMKYXXM” motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the “LHMKYXXM” motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the “LHMKYXXM” motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

Gene sequencing technology has been playing an important role in many aspects, such as life science, disease medicine and health medicine, particularly in the extremely tough process of fighting against 2019-novel coronavirus. Drawing DNA restriction map is a particularly important technology in genetic biology. The simplified partial digestion method (SPDP), a biological method, has been widely used to cut DNA molecules into DNA fragments and obtain the biological information of each fragment. In this work, we propose an algorithm based on 0–1 planning for the location of restriction sites on a DNA molecule, which is able to solve the problem of DNA fragment reconstruction just based on data of fragments’ length. Two specific examples are presented in detail. Furthermore, based on 1000 groups of original DNA sequences randomly generated, we define the coincidence rate and unique coincidence rate between the reconstructed DNA sequence and the original DNA sequence, and then analyze separately the effect of the number of fragments and the maximum length of DNA fragments on the coincidence rate and unique coincidence rate as defined. The effectiveness of the algorithm is proved. Besides, based on the existing optimization solution obtained, we simulate and discuss the influence of the error by computation method. It turns out that the error of position of one restriction site does not affect other restriction sites and errors of most restriction sites may lead to the failure of sequence reconstruction. Matlab 7.1 program is used to solve feasible solutions of the location of restriction sites, derive DNA fragment sequence and carry out the statistical analysis and error analysis. This paper focuses on basic computer algorithm implementation of rearrangement and sequencing rather than biochemical technology. The innovative application of the mathematical idea of 0–1 planning to DNA sequence mapping construction, to a certain extent, greatly simplifies the difficulty and complexity of calculation and accelerates the process of 'jigsaw' of DNA fragments. Supplementary Information The online version contains supplementary material available at 10.1007/s12539-021-00419-6.

The domestic Bactrian camel is a valuable livestock resource in arid desert areas. Therefore, it is essential to understand the roles of important genes responsible for its characteristics. We used restriction site-associated DNA sequencing (RAD-seq) to detect single nucleotide polymorphism (SNP) markers in seven domestic Bactrian camel populations. In total, 482,786 SNPs were genotyped. The pool of all remaining others were selected as the reference population, and the Nanjiang, Sunite, Alashan, Dongjiang, Beijiang, Qinghai, and Hexi camels were the target populations for selection signature analysis. We obtained 603, 494, 622, 624, 444, 588, and 762 selected genes, respectively, from members of the seven target populations. Gene Ontology classifications and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and the functions of these genes were further studied using Genecards to identify genes potentially related to the unique characteristics of the camel population, such as heat resistance and stress resistance. Across all populations, cellular process, single-organism process, and metabolic process were the most abundant biological process subcategories, whereas cell, cell part, and organelle were the most abundant cellular component subcategories. Binding and catalytic activity represented the main molecular functions. The selected genes in Alashan camels were mainly enriched in ubiquitin mediated proteolysis pathways, the selected genes in Beijiang camels were mainly enriched in MAPK signaling pathways, the selected genes in Dongjiang camels were mainly enriched in RNA transport pathways, the selected genes in Hexi camels were mainly enriched in endocytosis pathways, the selected genes in Nanjiang camels were mainly enriched in insulin signaling pathways, while the selected genes in Qinghai camels were mainly enriched in focal adhesion pathways; these selected genes in Sunite camels were mainly enriched in ribosome pathways. We also found that Nanjiang (HSPA4L and INTU), and Alashan camels (INO80E) harbored genes related to the environment and characteristics. These findings provide useful insights into the genes related to the unique characteristics of domestic Bactrian camels in China, and a basis for genomic resource development in this species.

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

International audience; Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3'-end of the forward primer was next to the polymorphic site, and the third base from the 3'-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

Taking advantage of “Y” junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a–S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization–cleavage–separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.

Toxoplasma gondii, an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates.

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

The standard binary enzymatic assembly, which operates by inserting one DNA fragment into a plasmid, has a higher assembly success rate than the polynary enzymatic assembly, which inserts two or more fragments into the plasmid. However, it often leaves a nucleotide scar at the junction site. When a large DNA molecule is assembled stepwise into a backbone plasmid in a random piecewise manner, the scars will damage the structure of the original DNA sequence in the final assembled plasmids. Here, we propose an in vitro Seamless Stack Enzymatic Assembly (SSEA) method, a novel binary enzymatic assembly method involving a seamless strategy of splicing restriction sites via a stepwise process of multiple enzymatic reactions that does not leave nucleotide scars at the junction sites. We have demonstrated the success and versatility of this method through the assembly of 1) a 4.98 kb DNA molecule in the 5′ → 3′ direction using BamHI to generate the sticky end of the assembly entrance, 2) a 7.09 kb DNA molecule in the 3′ → 5′ direction using SmaI to generate the blunt end of the assembly entrance, and 3) an 11.88 kb DNA molecule by changing the assembly entrance.

The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0-499 bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4-5 single cohesive end systems were recommended to digest the genome separately.

In Morocco, Leishmania infantum is the main etiologic agent of human and canine visceral leishmaniasis (VL). This species has been proven to be an opportunistic agent in HIV+ patients and is also responsible of sporadic cutaneous leishmaniasis (CL).This work aims to evaluate the genetic variability of Moroccan L. infantum strains based on PCR-RFLP analysis of the kinetoplastid DNA (kDNA) minicircles. A total of 75 DNA samples extracted from positive Giemsa-stained smears (n=32) and from L. infantum cultures (n=43) was studied. The samples have been taken from VL patients infected (n=7) or not (n=56) by HIV, patients with CL (n=2) and finally from infected dogs (n=10). An hypervariable region of kDNA was amplified using the primers MC1 and MC2; the PCR products were digested separately by a panel of nine restriction enzymes. The presence or absence of restriction fragments was scored in a binary matrix and the SplitsTree4 software was used for the construction of a Neighbor-Net network. Moroccan L. infantum population showed an important level of variability with the identification of 6 genotypes. For each genotype a PCR product was sequenced, confirming the presence of all the expected restriction sites. The predominant profile was the genotype B. A new genotype, named Q was detected for the first time, whereas the four other genotypes (G, K, N and O) were reported sporadically in the Mediterranean basin. The Neighbor-Net network segregates our L. infantum population into 3 clusters: Cluster I includes genotype B, cluster II grouping the genotypes O, Q and G and finally the cluster III contains the genotype N. The kDNA-PCR-RFLP assay is suitable for use directly on biological samples; it reveals an important degree of genetic variability among L. infantum strains even those belonging to the same zymodeme what is of great epidemiological interest.

In their recently corrected manuscript, "Breaking RAD: An evaluation of the utility of restriction site associated DNA sequencing for genome scans of adaptation", Lowry et al. argue that genome scans using RADseq will miss many loci under selection due to a combination of sparse marker density and low levels of linkage disequilibrium in most species. We agree that marker density and levels of LD are important considerations when designing a RADseq study; however, we dispute that RAD-based genome scans are as prone to failure as Lowry et al. suggest. Their arguments ignore the flexible nature of RADseq; the availability of different restriction enzymes and capacity for combining restriction enzymes ensures that a well-designed study should be able to generate enough markers for efficient genome coverage. We further believe that simplifying assumptions about linkage disequilibrium in their simulations are invalid in many species. Finally, it is important to note that the alternative methods proposed by Lowry et al. have limitations equal to or greater than RADseq. The wealth of studies with positive impactful findings that have used RAD genome scans instead supports the argument that properly conducted RAD genome scans are an effective method for gaining insight into ecology and evolution, particularly for non-model organisms and those with large or complex genomes.

Background Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Results Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Conclusions Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface (http://louiejtaylor.github.io/pyViKO/), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0920-3) contains supplementary material, which is available to authorized users.

Restriction-site associated DNA sequencing (RAD-seq) and target capture of specific genomic regions, such as ultraconserved elements (UCEs), are emerging as two of the most popular methods for phylogenomics using reduced-representation genomic data sets. These two methods were designed to target different evolutionary timescales: RAD-seq was designed for population-genomic level questions and UCEs for deeper phylogenetics. The utility of both data sets to infer phylogenies across a variety of taxonomic levels has not been adequately compared within the same taxonomic system. Additionally, the effects of uninformative gene trees on species tree analyses (for target capture data) have not been explored. Here, we utilize RAD-seq and UCE data to infer a phylogeny of the bird genus Piranga. The group has a range of divergence dates (0.5–6 myr), contains 11 recognized species, and lacks a resolved phylogeny. We compared two species tree methods for the RAD-seq data and six species tree methods for the UCE data. Additionally, in the UCE data, we analyzed a complete matrix as well as data sets with only highly informative loci. A complete matrix of 189 UCE loci with 10 or more parsimony informative (PI) sites, and an approximately 80% complete matrix of 1128 PI single-nucleotide polymorphisms (SNPs) (from RAD-seq) yield the same fully resolved phylogeny of Piranga. We inferred non-monophyletic relationships of Pirangalutea individuals, with all other a priori species identified as monophyletic. Finally, we found that species tree analyses that included predominantly uninformative gene trees provided strong support for different topologies, with consistent phylogenetic results when limiting species tree analyses to highly informative loci or only using less informative loci with concatenation or methods meant for SNPs alone.

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~4.7 mg L⁻¹. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.

A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO 4 and NH 2 groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3′ ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments.

The genera SPO1-like and Twort-like viruses in the subfamily Spounavirinae of the family Myoviridae have been newly proposed, with the reorganization of the SPO1-related bacteriophages (phages). A criterion defining these viral genera is the presence/absence of DNA modifications. In this study, liquid chromatography/mass spectrometry showed that phages ϕEF24C and K of the subfamily Spounavirinae have unmodified DNA, which classifies them as Twort-like viruses. Moreover, in the subfamily Spounavirinae, DNA modification and elimination of a particular DNA sequence were suggested to be the major antirestriction strategies of the SPO1-like and Twort-like viruses, respectively.

From our analysis of repeat sequences in the rye genome, the presence of multiple restriction sites of EcoO109I (5′–PuGGNCCPy–3′) across the genome has been predicted. By first using primers designed to contain EcoO109I sites in polymerase chain reaction (PCR), polymorphic DNA markers were effectively obtained. A total of 43 types of 10-mer primers containing EcoO109I sites were applied for PCR by using genomic DNA of Secale cereale self-fertile line IR27 and Triticum aestivum ‘Chinese Spring’ (CS) as the template. Twenty two primers detected polymorphisms between wheat and rye, and they were applied for PCR using a series of CS wheat – ‘Imperial’ rye chromosome addition lines as templates. Nine chromosome-specific amplification fragments identified on five chromosomes were collected from gels and hybridized with nylon membrane-transferred PCR products from the wheat–rye chromosome addition lines. The gel blot was only observed between the collected fragments; therefore, these fragments were confirmed to be chromosome-specific. These fragments were sequenced and converted to sequence-tagged site (STS) primers. We therefore introduce a new method for building chromosome-specific DNA markers: (i) multiple polymorphic fragments can be obtained from EcoO109I primers and (ii) the addition of three nucleotides to the EcoO109I site restricts the amplification region to generate chromosome-specific fragments.

The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356 bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204 bp. The 356 bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356 bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10.

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coli strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type.

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum , but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum , and the native promoter of sacB is weak in C. glutamicum . The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter P lacM and a novel vector pDXW-3 were constructed. P lacM is 18 times stronger than the native promoter of sacB in C. glutamicum . The pDXW-3 contains B. subtilis sacB with the P lacM fused at the 5′-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for Xho I, Not I, Eag I, Sal I, Sac I, Bam HI, and Nhe I, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB -based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.

The synthesis of complete genes is becoming a more and more popular approach in heterologous gene expression. Reasons for this are the decreasing prices and the numerous advantages in comparison to classic molecular cloning methods. Two of these advantages are the possibility to adapt the codon usage to the host organism and the option to introduce restriction enzyme target sites of choice. C.U.R.R.F. (Codon Usage regarding Restriction Finder) is a free Java®-based software program which is able to detect possible restriction sites in both coding and non-coding DNA sequences by introducing multiple silent or non-silent mutations, respectively. The deviation of an alternative sequence containing a desired restriction motive from the sequence with the optimal codon usage is considered during the search of potential restriction sites in coding DNA and mRNA sequences as well as protein sequences. C.U.R.R.F is available at http://www.zvm.tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/mikrobiologie/allgemeine_mikrobiologie/currf .

Purpose Characterisation of circulating melanoma cells (CMC) for BRAF status can provide a strategy for non-invasive serial genotype monitoring in patients receiving BRAF inhibitors. We aimed to establish a method for BRAF mutation analysis at CMC level and we applied it in a cohort of CMC-positive patients. Methods We established a sensitive method for detection of BRAF mutations at codon 600 in whole blood samples of patients with melanoma, positive for presence of CMC. The method based on selective cleavage of wild-type sequences by taking the advantage of the presence of a TspR1 enzyme restriction site located at the site of mutation. Results In a serial dilution experiment spiking BRAF mutated cDNA into BRAF wild-type cDNA the method allowed to detect mutated cDNA till a dilution of 1:10 4 . Peripheral blood (PB) samples resulting positive for CMC and matched tissue specimens from 21 different AJCC stage IV melanoma patients were analysed. A 91% (19/21) correspondence between BRAF status in tissue and PB specimens was observed. In a patient (whose melanoma showed to bear the BRAF V600E mutation in blood, but not at tissue level) our analysis showed that blood samples with PCR evidence for CMC were heterogeneous for BRAF status under limiting-dilution conditions, suggestive of heterogeneity of CMC. Conclusion The method reported here represents a rapid approach for determination of BRAF status in the blood of patients with CMC.

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.

The ATP-binding cassette transporter genes include the pleiotropic drug resistance (PDR) family found only in fungi and plants. These transporters transport toxic compounds across biological membranes. Here, we investigated the evolution of the PDR1 gene in Brachypodium distachyon, a widely distributed temperate grass species that belongs to the Poaceae (Gramineae) family, which also contains the domesticated cereal crops. Because this species has multiple ploidy levels, investigating PDR1 evolution in B. distachyon will offer insights into the formation and evolution of polyploidy. From 23 B. distachyon ecotypes, 39 PDR1 homologs were identified. All ecotypes had either one or two PDR1 copies. Based on restriction site analysis, the PDR1 homologs were classified as E or H type. All but one diploid and tetraploid ecotypes had only a single H type PDR1. All but one hexaploid ecotypes had both an E and a H type PDR1. Phylogenetic analysis revealed that each type formed a well-supported cluster. The two PDR1 types appeared to evolve differently. These different evolutionary patterns could indicate a difference in age between the two types or might indicate different mutation rates or selection pressures on the two types. The phylogenetic analysis also revealed that the hexaploid ecotypes shared a genomic origin for their E type PDR1, but there were multiple origins for hexaploid H type PDR1 homologs. Overall, the results suggest that tetraploid and hexaploid might be misnomers in B. distachyon and suggest a complex polyploidization history during B. distachyon evolution.

Here we describe a convenient and robust ligase-independent method for construction of combinatorial and random mutant libraries. The homologous genes flanked by plasmid-derived DNA sequences are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of flanking primers, followed by assembly of the chimeric genes and linearized vector by PCR to introduce recombinant plasmids of a combinatorial library. Commonly, it is difficult to find proper restriction sites during the construction of recombinant plasmids after DNA shuffling with multiple homologous genes. However, this disadvantage can be overcome by using the ligase-independent method because the steps of DNA digestion and ligation can be avoided during library construction. Similarly, DNA sequences with random mutations introduced by error-prone PCR can be used to construct recombinant plasmids of a random mutant library with this method. Additionally, this method can meet the needs of large and comprehensive DNA library construction.

The ability of 223 Type II restriction endonucleases to hydrolyze RNA–DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic or interrupted-palindromic DNA sequences, catalyze robust and specific cleavage of both RNA and DNA strands of such a substrate. Time-course analyses indicate that some endonucleases hydrolyze phosphodiester bonds in both strands simultaneously whereas others appear to catalyze sequential reactions in which either the DNA or RNA product accumulates more rapidly. Such strand-specific variation in cleavage susceptibility is both significant (up to orders of magnitude difference) and somewhat sequence dependent, notably in relation to the presence or absence of uracil residues in the RNA strand. Hybridization to DNA oligonucleotides that contain endonuclease recognition sites can be used to achieve targeted hydrolysis of extended RNA substrates produced by in vitro transcription. The ability to ‘restrict’ an RNA–DNA hybrid, albeit with a limited number of restriction endonucleases, provides a method whereby individual RNA molecules can be targeted for site-specific cleavage in vitro.

Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.

Study: Haplotypes linked to glucose-6-phosphate dehydrogenase (G6PD) genotypes were defined by studying six intragenic restriction fragment length polymorphisms (RFLPs) in 141 G6PD deficient and 252 normal chromosomes.Results: Only four of the 64 possible haplotypes were observed, indicating marked linkage disequilibrium. All the G6PD deficient mutations were associated with either haplotype I or VII, which are similar to the common G6PD B variant observed in the present study except the G6PD Namoru mutation which corresponded to mainly haplotype VIIa where a Nla III restriction site was created due to this mutation.Conclusion: The limited number and low haplotype diversity probably indicates a strong selective pressure on the G6PD gene.

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight-base-pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 A resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a betabetaalpha-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a 'vector modification fragment'. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose 'Highly Efficient gene Silencing Compatible vector' (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.

The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than approximately 10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae.

Artificial microRNA (amiRNA) is becoming a powerful tool for silencing genes in plants, and several amiRNA vectors have recently been developed based on the natural precursor structures of ath-miR159a, ath-miR164b, ath-miR172a, ath-miR319a and osa-miR528. In this study we generated a simple amiRNA vector (pAmiR169d) based on the structure of Arabidopsis miR169d precursor (pre-miR169d). Two unique restriction sites were created inside the stem region of pre-miR169d, which allows for the artificial miRNA sequences to be cloned as either ~80 bp synthetic oligonucleotides or PCR products. A beta-glucuronidase:green florescent protein fusion gene (GUS-GFP) was efficiently silenced in transient assays using a pAmiR169d-derived construct targeting the GUS-GFP sequence. 5'-RACE showed that the target GUS-GFP transcript was cleaved precisely at the expected position across nucleotides 10 and 11 of the artificial miRNA. Thus, pAmiR169d allows for both easy construction of artificial miRNA constructs and efficient silencing of target genes in plants.

A study of polymorphisms of mitochondrial DNA (mtDNA) of West African N'Dama (Bos taurus) and East African Zebu (B. indicus) cattle was carried out to obtain information on maternal phylogenetic relationships between these breeds. A relatively large sample size was made possible by using polymerase chain reaction (PCR) amplification of DNA prepared from small blood samples to generate fragments of two known polymorphic mtDNA regions, one within the gene encoding subunit 5 of NADH dehydrogenase and one encompassing the entire D-loop. This approach allowed us to achieve a higher resolution restriction analysis on mtDNA from more animals than would have been possible by conventional methods. PCR-amplified mtDNA of 58 animals from five populations was examined at 26 restriction sites by 16 enzymes. In this way 154 nucleotides of mtDNA were scanned for polymorphism. Six polymorphic sites were located by this means, five of which were within the D-loop and one of which was within the NADH dehydrogenase 5 gene. None of the polymorphisms observed could be considered typical of breed or type.

Summary We identified the HindIII restriction site polymorphism of K-casein in cattle reported by Pinder et al. (Animal Genetics 22, 11, 1991) and found an additonal polymorphism (RsaI) in cattle and bison. The HindIII and RsaI restriction sites were mapped and three haplotypes (alleles) were identified. Preliminary screening of 39 cattle and 71 bison revealed one allele restricted to cattle, one restricted to bison, and one shared by the species. No fixed allelic differences were observed among cattle breeds or among bison herds or subspecies.

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5'-CCGC/GG-3' ("/" marks the cleavage position) and cuts it to generate 3'-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.

The application of the peptide-linked beta2-microglobulin (beta2m) strategy is limited in some cases due to the incompatibility between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted to overcome this restriction. Three peptide-linked beta2m genes, HBc(18-27)-hbeta2m gene, OVA(257-264)-mbeta2m gene and HER2/neu(369-377)-mbeta2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked beta2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes.

As a widely used and expensive herbal medicine, Panax ginseng has many adulterants in the commercial market. PCR-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) based on 5S rDNA sequence analysis were applied to identify two common adulterants of P. ginseng. The sizes of 5S rRNA gene non-transcribed spacers (NTS) sequences in P. ginseng and its adulterants were determined, ranging from 143 to 424 bp. The PCR product of P. ginseng only could be digested among the tested specimens because of its specific SpeI restriction site found in the 5S rDNA sequence. In addition, P. ginseng was successfully identified from compound medicinal preparations and from the Single-Taste medicines. These results suggest that the methods are able to authenticate P. ginseng.

To construct a vector for caspase-1 independent expression of canine IL-18, the signal sequence of canine IL-12p40 was fused to the sequence of mature IL-18 on the NdeI restriction site which is located at the 3′ end of the signal sequence. The resulting vector expressed coding protein from transfected mammalian cells. The expressed protein was shown to have IL-18 bioactivity in a INF-γ-inducing assay. These results suggest that the expression vector is the desired tool for advancement of dendritic cell (DC)-based cancer therapy, provided that the vector can successfully be transfected into dendritic cells. We propose a simple and widely applicable method for providing the signal sequence.

The merozoite surface antigen-2 (msa-2) family of Babesia bovis is a group of variable genes that share conserved 5' and 3' ends and encode for membrane-anchored glycoproteins that have been postulated as vaccine candidates. In this work, we analyzed the sequences of three of these genes (msa-2a1, a2, and 2b) from two geographically distant strains and detected a certain degree of genotypic diversity that could be exploited to work out new molecular tools for the discrimination of B. bovis field samples. Here we describe a PCR restriction assay that was developed based on this observation and tested on several B. bovis strains and isolates. The results show a strain-specific band pattern in geographically distant isolates, indicating the presence of differentially located BspMI restriction sites. This approach provides a simple method for the differentiation of American B. bovis strains.

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t w2 , t 0 , t w32 , t w71 , and t w75 . We show that only t w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t w71 and t w75 mice.

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.