PCR Restriction Enzyme Digestion Research Articles

Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana

BackgroundTo date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system.ResultsProtoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems.ConclusionsThe PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Preimplantation Genetic Testing of Achondroplasia by Two Haplotyping Systems: Short Tandem Repeats and Single Nucleotide Polymorphism

Here, we present the successful application of two different preimplantation genetic testing for monogenic diseases (PGT-M) methods for a couple facing the genetic risk of Achondroplasia (ACH). The first preimplantation genetic haplotyping (PGH) cycle was based on short tandem repeats (STRs) and 8 STRs were chosen. The multiple displacement amplification (MDA) products were analyzed using the informative STR loci and PCR-restriction enzyme digestion of FGFR3. A healthy girl was delivered. Two years later, we performed the second PGT-M cycle for this couple with a newly established PGT-M platform based on next generation sequencing (NGS). Haplotype analysis was established by a selection of several informative single nucleotide polymorphisms (SNPs). Preimplantation genetic testing for aneuploidy (PGT-A) was also performed on embryos with normal FGFR3 genotype. Another healthy girl was born. PGH system could be established using STRs or NGS-SNP systems. The NGS-SNP system could detect more sites and simultaneously performs PGT-A with an automated operation.

Comment on: Barker et al. (2008) Two Single Nucleotide Polymorphisms Identify the Highest-Risk Diabetes HLA Genotype: Diabetes 57:3152–3155, 2008

Barker et al. (1) recently proposed two single nucleotide polymorphisms (SNPs) to identify the highest HLA risk for type 1 diabetes. The SNP rs7454108, genotyped by TaqMan probes, tags the HLA haplotype DR4-DQA1\*0301-DQB1\*0302 (DR4-DQ8), whereas rs2040410, genotyped using PCR–restriction enzyme digestion, tags DR3-DQA1\*0501-DQB1\*0201 (DR3-DQ2.5). The initial role of HLA in type 1 diabetes was indicated by association with the HLA-DR3 and -DR4 antigens, but, more recently, the HLA-DQA1 and - DQB1 genes were shown to be …

Molecular Diagnostics of Porcine Stress Syndrome Susceptibility Associated with the Arg615Cys Mutation Using Real-Time PCR with Fluorescent Hybridization Probes

ObjectiveThe purpose of the present study was to develop a molecular genotyping method test by using a real time PCR hybridization probe and applying it to the analysis of C1843T mutations of the Sus scrofa RYR1 gene.Animals population Three PSS-susceptible and PSS non-susceptible crossbred swine races were used for the experiments: Pietrain X Landrace Belga, Pietrain X Large White and Pietrain X Duroc. MethodsWe have developed a genotyping method by using a hybridization probe and applied it to the analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results obtained by hybridization probe strategies were confirmed by restriction analysis and sequencing. In addition, phenotype/genotype correlation analyses were developed by using the in vitro contracture test and confirmed the in vivo halothane-succinylcholine challenge. ResultsThe real-time PCR with fluorescent hybridization probe methodology was designed to identify homozygous PSS-resistant, PSS-susceptible animals as well as heterozygous carriers. All cases genotyped by fluorescent hybridization probes were in agreement with PCR restriction enzyme digestion and sequencing and showed a 100% concordance between the in vivo and in vitro porcine stress syndrome (PSS) susceptibility results. Conclusions and clinical relevanceThe real-time PCR with fluorescent hybridization probe method described here provides a rapid, easily interpretable and reliable tool for genotyping the C1843T (Arg615Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of PSS-susceptibility and genetic selection.

Single-Nucleotide-Polymorphism Typing and Genetic Relationships of Salmonella enterica Serovar Typhi Isolates

Salmonella enterica serovar Typhi is a clone with a low level of variation. We developed a molecular typing method for serovar Typhi using 38 genome-wide single-nucleotide polymorphisms (SNPs) as markers detected by PCR-restriction enzyme digestion. The 73 worldwide serovar Typhi isolates studied were separated into 23 SNP profiles and four distinct genetic groups. Serovar Typhi isolates expressing the unique flagellar antigen z66 were found to cluster together and branch off from the ancestral group, suggesting that serovar Typhi was initially monophasic with only an H1 antigen and subsequently gained the z66 antigen. Typing using the 38 SNPs gave a discriminatory power of 0.87, and a minimum of 16 SNPs may be used to achieve the same level of differentiation. The SNP typing method we developed will be a valuable tool for global epidemiology studies of serovar Typhi.

Detection of two novel mutations of iduronate-2-sulfatase gene in pa-tients with mucopolysaccharidosis typeⅡ

In the present study, through PCR amplification and direct sequencing of mutation "hotspots", we were able to identify two novel mutations in the human iduronate-2-sulfatase (IDS) gene in two patients from unrelated families with mucopolysaccharidosis type II(MPS II). The novel mutation IVS 6 -1g-->a affected the 3' splice acceptor site of intron 6, and was predicted to result in exon skipping. The novel mutation c.1587-1588 ins T involved a single base insertion be-tween nucleotides 1,587 and 1,588 in exon 9, and was predicted to result in frame shift and premature termination. The two novel mutations did not occur in 6 other unrelated MPS patients or in 100 alleles from normal individuals, indicating that they were not polymorphisms. The PCR-restriction enzyme digestion showed that the two newly identified mutations were of maternal origin, which was consistent with the X-linked recessive disorder. These findings suggest that the IDS gene mutations could be detected by amplifying mutation "hotspots", direct sequencing and restriction digestion analysis, and the newly identified mutations may be disease-causing.

Neurofilament M gene in a French-Canadian Population with Parkinson’s Disease

Recently, a single base pair substitution (G1747A) mutation of the neurofilament M (NF-M) gene was reported in a French-Canadian patient with early onset Parkinson's disease (PD). Three unaffected siblings were found to be heterozygotes for the NF-M Gly336Ser mutation but, to date, no other affected PD individuals have been found with a similar mutation. No other individuals with Parkinson's disease and of similar ethnic background have been screened for this mutation. We screened 102 French-Canadian patients with definite PD and 45 French-Canadian controls for this substitution in the NF-M gene using a PCR-restriction enzyme digestion method. None of the patients or controls carried this mutation. Our results would indicate that this mutation is not common even in a PD population of similar ethnic background and suggest this change represents a rare variant. However, these results do not exclude the possibility that other mutations in this gene could be present.

Diagnosis of Hunter syndrome: identification of iduronate-2-sulfatase gene mutations in patients with mucopolysaccharidosis type II in Chinese

Objective: Mucopolysaccharidosis type II (Hunter syndrome) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS). Mutations of the IDS gene which is located on Xq27.3-q28 have been identified as responsible of Hunter syndrome or mucopolysaccharidosis type II. We’d like to investigate and establish the gene diagnosis methods for IDS gene in mucopolysaccharidosis type II patients. Design: Obtain the genomic DNA of patients and his parents, amplify with PCR, and analyse the PCR product with PCR-SSCP. Directly sequence the PCR product with abnomal band in PCR-SSCP. At last restriction enzyme digestions are used to verlify the result of DNA sequencing. Materials and Methods: Determination of carrier status in families, affected by this disorder has been performed using a number of biochemical tests. PCR amplification on genomic DNA of patients and his parents. PCR-SSCP analysis was applied to detect the mutations of exons which were amplificated in PCR. DNA sequencing and PCR-restriction enzyme digestion were applied to analyze the mutation which has been detect by PCR-SSCP. computer programs were necessary to elucidate the nature of the mutations. Results: Abnormal band in exon 7 and 9 of the IDS gene were found by applying PCR-SSCP; Two new alterations at the IDS locus, the mutation 1253 GμT of exon7 and the mutation 1672 CμT of exon9, were found in the two patients through DNA sequencing, The PCR-restriction enzyme digestion showed that enzyme digestion locations changed in the patients and their mothers, Which verified the results of sequencing analysis. Conclusion: To date, none of the available enzymatic tests have been shown to identify accurately 100% of obligate carriers. DNA testing provides an opportunity to identify carriers unambiguously. Combined applications of PCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion can screen and diagnose MPS a effectively, quickly and perform easily. And information of detected mutations are potentially useful for the genotype-phenotype correlation, prognosis, possible therapeutic intervention, prenatal diagnosis and preimplantation genetic diagnosis of the pedigree.

Novel missense mutation of the UGT1A1 gene in Thai siblings with Gilbert's syndrome

Gilbert's syndrome is a common inherited disorder of bilirubin metabolism contributing to the development of neonatal jaundice and causing recurrent jaundice after the neonatal period. In the patients with Gilbert's syndrome, mutations have been reported in the promoter and exons of the uridine diphosphate-glucuronosyl transferase 1 (UGT1A1) gene on chromsome 2q37, which encodes bilirubin uridine diphosphate-glucuronosyltransferase. However, the genetic basis of Gilbert's syndrome, including its inheritance trait, remains to be clarified. Patients 1 and 2 were Thai sisters with Gilbert's syndrome. They had a history of prolonged neonatal jaundice and showed recurrent jaundice after their infancy, while the parents showed no symptoms. To search for the mutation in the patients, all exons of the UGT1A1 gene were amplified by polymerase chain reaction (PCR) and sequenced directly. The frequency of the mutation in controls was studied by PCR-restriction enzyme digestion method. The patients were homozygous for a novel single transition of T to C at nucleotide position 247 (exon 1), which would predict a substitution of leucine for phenylalanine at codon 83 of the enzyme protein. No other mutation was detected in any regions except exon 1. The parents with no symptoms showed heterozygosity for the mutation. Among the 110 Japanese controls, no homozygous individuals and three heterozygous individuals for the mutation were identified, giving a mutated allele frequency of 0.0136. A novel missense mutation in the UGT1A1 gene was identified in two Thai siblings with Gilbert's syndrome. The affected family showed that homozygosity for the mutation may lead to apparent symptoms and that the syndrome was inherited as an autosomal recessive trait. The mutation does not explain a high incidence of neonatal jaundice in Japan, because it is very rare in the Japanese population.

A molecular genetic analysis of the Kidd blood group polymorphism in Taiwanese, Thais, and Filipinos.

In this study, we molecularly detect the Jka/Jkb polymorphism in Taiwanese, Thais, and Filipinos with PCR-restriction enzyme digestion. We designed the primers with mutagenic bases to create two restriction enzyme cutting sites (EcoN I and Xba I) simultaneously to confirm the genetic polymorphism of Jka/Jkb. One hundred and seven unrelated Taiwanese, 107 Thais foreign laborers, and 102 Filipino foreign laborers in Taiwan were analyzed. The frequency of Jka and Jkb is 0.51 and 0.49 in Taiwanese, 0.44 and 0.56 in Thais, and 0.45 and 0.55 in Filipinos, respectively. The molecular analysis of Jka/Jkb polymorphism is simple, accurate and could confirm the uncertain results obtained by serological typing. The method could be used for predicting fetal Kidd genotype from amniocyte DNA, and to avoid the hemolytic disease of newborns caused by Jk antibody.