Amplification Of Genomic DNA Fragments Research Articles

An Efficient Method for DNA Purification‐Free PCR from Plant Tissue

Amplification of genomic DNA fragments by PCR is necessary for plant molecular biology approaches such as genotyping. While this is a routine molecular technique in a modern laboratory, there are still significant hurdles when analyzing a large number of samples or collecting and storing samples while in the field. Because PCR amplification directly from plant tissue is often unsuccessful due to various inhibitors, genomic DNA purification is usually required, which involves laborious and time-consuming procedures or costly materials, particularly when using commercial kits. These undermine scalability and use in less-equipped settings. In addition, plant tissues and purified DNA need to be stored under proper conditions to avoid degradation. Here, we describe a low-cost, high-throughput PCR method to amplify genomic DNA fragments from plant tissue pounded to cellulose-based filter paper without the need for DNA purification or special equipment for sample storage. In this protocol, a small punch of plant tissue is pounded to a commercially available or homemade DNA storage card and directly placed into a PCR mixture containing Tween-20, a non-ionic detergent, directly followed by PCR. We also describe the steps to prepare a homemade DNA storage card, which is easy to make and can be stored with plant tissue at room temperature for a long time without any special equipment, allowing us to test the same sample multiple times. We have used this method in at least eleven plant species, including arabidopsis, tomato, soybean, potato, cotton, and rice. Altogether, our method decreases labor and cost, thereby increasing throughput and making plant DNA-based molecular diagnostic assays accessible to resource-limited settings, including classrooms, and facilitating sample collection in the field. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Making a homemade cellulose-based DNA storage card Basic Protocol 2: Pounding plant tissue on a DNA storage card Basic Protocol 3: DNA-purification free PCR.

Study of Genetic Diversity of Glycyrrizha glabra L. Populations Using ISSR Molecular Markers

Twenty-two G. glabra populations were used to study the genetic diversity of ISSR molecular markers. 12 primers were used to amplification of genomic DNA fragments of G. glabra populations. High genetic diversity based on ISSR markers was observed among individuals. A total of 130 bands were formed and 105 bands were polymorphic. The mean polymorphism percentage among studied populations was 80.47. The highest polymorphic percentages were assigned to IS23, IS21, IS9, IS13 and IS15 primers. The mean of PIC and MI were 0.347 and 2.47, respectively. The Shannon index (I) varied between 0.207-0.393 and the Nei genetic variation index (h) from 0.140 to 0.026. Darab and Solataniyeh populations showed the lowest and highest genetic diversity, respectively. The percentage of polymorphic loci was varied between 35.224 to 65.71%. The observe allele number and effective alleles number was 1.46 and 1.34, respectively. Based on the genetic distance Nei, populations Bardsir and Baft had the highest genetic similarity (0.888) and populations Bardsir and Solataniyeh had the least genetic similarity (0.132). The studied populations were grouped into three main groups by cluster analysis using UPGAM and Jaccard's similarity coefficient. The results showed that the ISSR marker is a reliable marker system for revealing a high level of polymorphism and can be used to study genetic diversity and further examinations as a subset of breeding programs in G. glabra.

Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

To determine exon/intron organization of the Toxocara canis (T.canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25°C for the forward reaction (phosphagen synthesis). Arginine kinase in T.canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542bp to 2 500bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The Km value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The Km value of the mutant (Serine to Glycine) increased to 0.19mM. The Km value (0.19mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A.indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A.indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A.indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T.canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T.canis.

Chlamydomonas (Chlorophyceae) colony PCR

The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.

Identification of Novel Single‐Nucleotide Polymorphisms in Chinook Salmon and Variation among Life History Types

AbstractSingle‐nucleotide polymorphisms (SNPs) are simple base substitutions or small indels in otherwise conserved regions of nuclear or mitochondrial DNA. Assays detecting these genetic markers have been a powerful tool for conserving and managing fish populations. In nonmodel species, however, available DNA sequence data are limited and inventive techniques must be employed in screening for SNPs. In this study, we used expressed sequence tags from rainbow trout Oncorhynchus mykiss to design primers for amplification of genomic DNA fragments from Chinook salmon O. tshawytscha. The regions flanking the repeat sequence of published microsatellite loci in Chinook salmon were also chosen to examine nucleotide variation. Thirty‐two individuals representing the majority of the species' range from Russia to California were sequenced at 19 loci to identify SNPs. We discovered 58 polymorphisms during the screen and chose 13 for development into 5′ exonuclease assays (TaqMan assays). The genotypes from the TaqMan assays were compared with sequence data from individuals from the ascertainment panel to confirm proper allele designations. To evaluate the power of these markers to differentiate populations and life history types, we typed 91 individuals representing the major lineages of Chinook salmon with 11 validated assays. Significant allele frequency differences were observed among life history types of Chinook salmon at seven SNP loci, demonstrating the power of these markers to differentiate lineages.

Linear chromosomes in bacteria: no straight edge advantage?

The beginning of 2007 brought us the complete genome of the yeast Pichia stipitis, five archaeal and more than 25 completely sequenced bacterial genomes (Table 1). In addition, there were two genomics-related papers that deserve a special discussion. One of them (Fuchs et al., 2007) described an unfinished genome of the ubiquitous marine phototrophic bacterium Congregibacter litoralis, while the other (Cui et al., 2007) examined the properties of the Escherichia coli K-12 strains with a linear chromosome.

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon

A cDNA fragment of the T cell receptor (TCR) α chain mRNA in Atlantic salmon ( Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR α chain constant region (Cα) gene and flanking regions was isolated and partially sequenced. The Cα gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Cα gene were found nearby the intact gene. One J segment was found 1.5 kb upstream of the Cα gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Cα junction. Representatives of five Vα gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Cα fragments from another individual revealed a slightly different Cα gene which most likely represents an allelic variant.

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon

A cDNA fragment of the T cell receptor (TCR) α chain mRNA in Atlantic salmon (Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR α chain constant region (Cα) gene and flanking regions was isolated and partially sequenced. The Cα gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Cα gene were found nearby the intact gene. One J segment was found 1.5 kb upstream of the Cα gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Cα junction. Representatives of five Vα gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Cα fragments from another individual revealed a slightly different Cα gene which most likely represents an allelic variant.

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.

Numerous, robust genetic markers for Plasmodium chabaudi by the method of amplified fragment length polymorphism

We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur™ filters. This reduced nucleated host cell contamination to around 1–10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8×10 −3 and 4.6×10 −4 per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory.

Amplification of genomic DNA fragments of Sarcocystis muris (Apicomplexa) cyst merozoites encoding a thiol (cysteine) proteinase.

Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen.

RAPD Fingerprinting of Chlorobium Strains

Summary In vitro amplification of genomic DNA fragments with single primers of arbitrary sequence was used as a rapid and sensitive method to obtain fingerprints of fourteen strains belonging to five Chlorobium species. Each strain showed a distinctive pattern of bands that permitted its unequivocal identification when the primer gyr lower was used. Our results indicate that the random amplified polymorphic DNA (RAPD) technique is a simple, fast and useful tool to complement the identification of newly isolated Chlorobium strains. Moreover, the cladistic analysis of thirteen Chlorobium strains was obtained by joining the data from pulsed field (PFGE) macrorestriction patterns, ribotyping and random amplified DNA (RAPD). The resulting distribution of strains shows some differences between the current classification within this genus and the distribution based on genetic relationships. Thus, taking into account molecular and phenotypic data seems to be the best way to have a better understanding of the taxonomy of these bacteria.