Restriction Endonuclease Cleavage Sites Research Articles

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction.

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal. Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment. The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

Allosteric Regulation of Aptamer Affinity through Mechano-Chemical Coupling.

The capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial-and-error testing or require prior knowledge of an aptamer's structure and ligand-binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double-stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin-binding aptamer and show that we can alter its affinity by as much as 65-fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely-tuned affinity modulation. This strategy requires no prior knowledge of the aptamer's structure and binding mechanism and should thus be generalizable across aptamers.

Allosteric Regulation of Aptamer Affinity through Mechano‐Chemical Coupling**

AbstractThe capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial‐and‐error testing or require prior knowledge of an aptamer's structure and ligand‐binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double‐stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin‐binding aptamer and show that we can alter its affinity by as much as 65‐fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely‐tuned affinity modulation. This strategy requires no prior knowledge of the aptamer's structure and binding mechanism and should thus be generalizable across aptamers.

Development of multiplex loop-mediated isothermal amplification for three foodborne pathogens

Staphylococcus aureus, Salmonella, and Shigella are three major foodborne pathogenic microorganisms that cause global public health problems. We developed a multiplex loop-mediated isothermal amplification (mLAMP) assay for simultaneous detection of S. aureus nuc, Salmonella fimY, and Shigella ipaH in fresh fruit juice using three sets of primers. In addition, three different restriction enzyme cleavage sites were designed in each forward inner primer (FIP), namely, XhoI in nuc FIP, KpnI in fimY FIP, and BamHI in ipaH FIP. DNA was amplified using the LAMP assay at 64 °C for 50 min followed by endonuclease restriction digestion to separate the LAMP products of three pathogens. The minimum amount of genomic DNA of S. aureus, Salmonella, and Shigella that could be detected by mLAMP was 100 fg/25 μL, whereas for mPCR, it was 1 pg/25 μL. The artificially contaminated juice can also be detected by mLAMP after enrichment, which had the limit of detection (LOD) of 2 CFU/10 mL. In conclusion, the mLAMP developed in this study could be potentially used in the detection of S. aureus, Salmonella, and Shigella in food, particularly as a primary screening method in developing areas.

A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression

BackgroundCell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively.ResultsHere, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies.ConclusionsThe new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

Protein expression and gene editing in monocots using foxtail mosaic virus vectors.

Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii.

BackgroundMutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii.MethodsBased on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.ResultsPCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain.ConclusionsThis assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.

Standardization and application of a modified RFLP-PCR methodology for analysis of polymorphisms linked to treatment resistance in Ancylostoma braziliense

BackgroundSingle nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the beta-tubulin isotype 1 gene are associated with benzimidazoles resistance in many helminths. Codon 167 mutation has never been described in hookworms; however, polymorphisms in codons 198 and 200 have been described for Ancylostoma caninum and Necator americanus. These mutations have never been investigated in Ancylostoma braziliense; therefore, it is not known if they are present in this species and whether they are correlated with treatment resistance. The RFLP-PCR technique has been used to analyze these polymorphisms in some nematodes, but depending on the species, these alterations do not create or eliminate any restriction enzyme cleavage site, making it impossible to use this technique. Here, we describe the standardization and application of a modified RFLP-PCR technique for detecting polymorphisms in individual A. braziliense worms recovered from naturally infected dogs in two Brazilian states.ResultsThe molecular techniques used were sensitive, specific, and easy to apply. To our knowledge, we report for the first time the presence of a polymorphism at codon 198 of the beta-tubulin gene of A. braziliense (1/81; 95% CI: 0–3.69%).ConclusionsIt is not known whether the presence of the mutation in codon 198 of the beta-tubulin gene of A. braziliense has importance for this parasite. However, based on studies of other helminths, it is possible that this polymorphism is directly related to the resistance to benzimidazoles. This may be a major concern, since this nematode has considerable relevance as a parasite of canids and felids and as one of the agents of cutaneous larva migrans in humans. Standardized methodologies will be useful for screening for polymorphisms in the beta-tubulin gene of canine hookworms in a broader population. The method could also be adapted for the analysis of other SNPs in other nematode species.

DNA Garden: A Simple Method for Producing Arrays of Stretchable DNA for Single-Molecule Fluorescence Imaging of DNA-Binding Proteins

Abstract We developed a simple method for producing arrays of stretchable DNAs, called DNA garden, for single-molecule fluorescence measurements. The method is based on microcontact printing of biotinylated bovine serum albumin (biotin-BSA) on a coverslip coated by 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and on the subsequent tethering of neutravidin and biotinylated DNA. Without the need for a microfabricated substrate used for DNA tethering, it facilitates single-molecule investigations of DNA and DNA-binding proteins based on fluorescence microscopic imaging. The salient advantage of DNA garden is continuous observation of DNA in the repeated cycles of extension and relaxation by flow control, enabling the characterization of processes occurring in and on the relaxed DNA. The DNA garden was applied to the detection of cleavage sites of restriction enzymes and for the observation of the sliding dynamics of a tumor suppressor, p53, along extended DNA at the single-molecule level. Furthermore, experiments based on the repetitive extension and relaxation of DNA demonstrated that p53 causes looping of DNA, probably by connecting multiple regions of the relaxed DNA. The DNA garden is expected to be a powerful tool for the single-molecule imaging assay of DNA and DNA-binding proteins.

“De-novo” amino acid sequence elucidation of protein G′e by combined “Top-Down” and “Bottom-Up” mass spectrometry

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

Identification of Regulatory-RNAs for Alternative Splicing of Ron Proto-Oncogene.

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.

Single base resolution analysis of 5-methylcytosine and 5-hydroxymethylcytosine by RRBS and TAB-RRBS.

Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5'CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-hydroxymethylcytosine (TAB-RRBS).

Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum

A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 103 times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys.

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species.

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5′-T/TAA-3′ in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Role of Bovine Bcl2A1 Gene in Staurosporine-Induced Apoptotic Cell Lines

Bcl2A1 (B-cell lymphoma 2 - related protein A1) is a member of a gene set that plays a critical role in regulating apoptosis of mammalian cells. The family is divided into 2 groups: (1) anti-apoptotic genes Bcl-2, Bcl-xL, Bcl2A1 and (2) pro-apoptotic genes Bid, Bax, Bak. Apoptosis in cells is regulated by a balanced expression of these groups. In this study, the ORF encoding the 175 amino acid bovine Bcl2A1 protein was amplified by reverse transcription polymerase chain reaction (RT-PCR), using specific primers deduced from the bovine Bcl2A1 mRNA sequence (www//ncbi.nlm.nih.org. Acc: AB195549) and which also contained appropriate restriction endonuclease cleavage sites for cloning. The product was then cloned into plasmid pcDNA3+ to construct the eucaryotic expression plasmid pcDNA3.Bcl2A1. Hela, L11, Vero and WSL cell lines were used to investigate apoptosis induced by staurosporine. Monitoring of cellular DNA fragmentation revealed that incubation with staurosporine at a concentration of 2 µM results in a sufficient level of apoptosis induction in Hela, L11, WSL cell cultures after 6 hours and in Vero cell cultures after 12 hours. Transfection of these cell lines with pcDNA3.Bcl2A1, using pseudorabies virus Us3 protein kinase (PrVUs3) which prevents staurosporine induced apoptosis by interacting with the pro-apoptotic proteins Bid and Bad as a control, proved that expression of the bovine Bcl2A1 gene blocks staurosporine-induced apoptosis in Hela and L11 cell lines. However, this activity was not observed in WSL and Vero cell cultures.

Restriction Map For NK104-NK105 of Nonrepeating Region For tcdB Toxin Gene in Clostridium difficile

This study was carried out for detected of the restriction map of tcdB gene for A - B + isolates were isolated from patients with a colitis disease in General Basrah Hospital by (AluI, DraI, ScaI) restriction enzymes.The genetic restriction map was detected by determining the locations of a number of restriction endonuclease cleavage sites on the DNA relative to each other. AluI enzyme was showed three bands (100 bp, 90 bp and 60 bp) revealing there are two sites of this enzyme, on the other hand DraI, ScaI gave two bands (125 bp) for each enzyme revealing there was a single site of these enzymes, A variety of tcdB gene which detected in this study a new modification increasing the virulence of A - B + isolates of C. difficile with the large defect of tcdA gene.

SC*994C>T causes the Scnull phenotype in Pacific Islanders and successful transfusion of Sc3+ blood to a patient with anti-Sc3

Abstract Antigens in the SC blood group system are expressed by the human erythrocyte membrane-associated protein (ERMAP). Two molecular bases have been reported for the Scnullphenotype: SC*307del2and SC*994C>T.We report our investigation of the molecular background of five Scnullindividuals from the Pacific Islands and describe the successful transfusion of Sc3+ blood to a patient with anti-Sc3 in her plasma. SC (ERMAP)exons 2, 3, and 12 and their flanking intronic regions were analyzed. The SC*994C>Tchange introduces a restriction enzyme cleavage site for Tsp45I, and polymerase chain reaction (PCR) products from exon 12 were subjected to this PCR–restriction fragment length polymorphism (RFLP) assay. The five samples had the variant SC*994T/T.One sample, from a first cousin of one Marshallese proband, was heterozygous for SC*1514C/T(in the 3′ untranslated region); the other four samples were SC*1514C/C(consensus sequence). Samples from white donors (n = 100) and African American donors (n = 99) were tested using the Tsp45I PCR-RFLP assay; all gave a banding pattern that was consistent with the SC*994C/Cconsensus sequence. In all five samples, our analyses showed homozygosity for the nonsense nucleotide change SC*994C>Tin an allele carrying the nucleotide associated with Sc1. Further investigation determined that one of the probands reported previously with the SC*994C>Tchange was from the Marshall Islands (which form part of the Micronesian Pacific Islands) and the other was from an unspecified location within the large collection of Pacific Islands. Taken together, the five known probands with the SC*994C>Tsilencing nucleotide change were from the Pacific Islands. Immunohematology 2013;29:69–72.

Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.

Rapid identification of Amaranthus caudatus and Amaranthus hypochondriacus by sequencing and PCR–RFLP analysis of two starch synthase genes

The objective of this study was to develop a PCR-RFLP method to identity the cultivated species of grain amaranth based on variations in the sequences of their starch synthase genes. We sequenced the SSSI and GBSSI loci in 126 accessions of cultivated grain amaranth collected from diverse locations around the world. We aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. Our analyses indicated that EcoRI would recognize the sequence 5'-GAATT/C-3' in the SSSI gene from Amaranthus caudatus L., and TaqI would recognize the sequence 5'-T/CGA-3' in the GBSSI gene from Amaranthus hypochondriacus L. The PCR products obtained using gene-specific primers were 423 bp (SSSI) and 627 or 635 bp (GBSSI) in length. These products were cut with different restriction enzymes resulting in species-specific RFLP patterns that could be used to distinguish among the cultivated grain amaranths. The results clearly showed that A. caudatus and A. hypochondriacus were easily differentiated at the species level using this method. Therefore, the PCR-RFLP method targeting amaranth starch synthase genes is simple and rapid, and it will be a useful tool for the identification of cultivated species of grain amaranth.