The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

Identification of multiple strains of Porphyromonas gingivalis using heteroduplex polymerase chain reaction in varying severity of chronic periodontitis

Aim: Research has demonstrated that there are multiple strains of Porphyromonas gingivalis with varying potency to cause periodontal disease. The current study aims at using heteroduplex polymerase chain reaction (PCR) to detect the strain diversity of P. gingivalis in periodontitis lesions of varying severity in a sample of the Indian population. Materials and Methods: Subgingival plaque samples were collected from 60 individuals with varying severity of chronic periodontitis and 30 individuals with a clinically healthy periodontium. The samples were subjected to PCR analysis to identify P. gingivalis, followed by heteroduplex analysis to identify the strain diversity in a given sample. Bacterial culture was carried out as a comparative standard. Results: Of the 56 samples that were positive for P. gingivalis by PCR, 54 samples yielded eight different heteroduplex patterns. Analysis of these patterns indicated that two strains of P. gingivalis were present in 41 individuals (45.6%) and three strains were present in 13 individuals (14.4%). Detection of P. gingivalis by PCR was significantly more in the periodontitis group as compared to the healthy group. Conclusions: Species-specific PCR and heteroduplex analysis provide a simple and accurate method to analyse the strain diversity of P. gingivalis. P. gingivalis was detected in both healthy periodontal sites as well as sites with periodontitis. The presence of two or three P. gingivalis strains was seen in 60% of the samples.

Application of heteroduplex mobility assay in quick identification of adenovirus subtypes in epidemic keratoconjunctivitis / 中华实验眼科杂志

Background The commonly used method of typing the adenovirus (AdV) for epidemic keratoconjunctivitis is direct DNA sequence.However,heteroduplex mobility assay (HMA) is found to be a faster method to identify the subtypes of adenovirus and is more conforming to the principle of cost and economic benefit.There are few studies to illustrate the application of HMA in epidemic keratoconjunctivitis.Objective This study analyzed the conserved region of hexon coding sequence and compared the outcomes between direct DNA sequence and HMA for classification of adenovirus in epidemic keratoconjunctivitis patients.The validity of HMA is evaluated by comparing the result of both studies Methods Two hundreds and fourteen patients with suspicious epidemic keratoconjunctivitis were included in Shanghai Eye Disease Prevention and Treatment Center or the clinical sites supervised by the Shanghai Prevention and Monitoring Office of Acute Hemerragic Conjunctivitis from January 2010 to December 2012.Sacconjunctival swab samples were collected from each patient under the informed consent.DNA of pathogens was extracted from the samples using QIA-amp minikit,and the conserved sequence with 366 bp at hexon protein coding region was amplified by PCR and sequenced subsequently to determine the infected adenovirus and their subtypes.These samples were simultaneously assayed by HMA,and the outcomes between DNA sequence and HMA were compared.Results Extracted DNA presented a yellow fluorescence band with the fragment size 35 kb and absorbance ratio at the wavelength 260 nm and 280 nm (A260/A280) was I.7.In the 214 samples,AdV type 1 (AdV1) was found in 4 samples,AdV2 in 33 samples,AdV3 in 15 samples,AdV4 in 12 samples,AdV8 in 19 samples,AdV19 in 15 samples and AdV37 in 8 samples.HMA showed the same outcome for the identification of AdV1,AdV2,AdV3,AdV8,AdV19 and AdV37 with direct DNA sequence.AdV4 was not feasible to HMA owing to 59.6％ (over 10％) mutation sites.Conclusions Direct DNA sequencing for conserved regions in coding sequence of hexon is an important way to identify causing-disease adenovirus subtypes in the patients with epidemic keratoconjunctivitis.HMA can offer a consistent result with the DNA sequencing,and it might be used as a suitable tool for large-scale molecular epidemiology researches.

Study of HIV-1 subtypes in serodiscordant couples attending an integrated counselling and testing centre in Mumbai using heteroduplex mobility analysis and DNA sequencing.

Aims: To determine the prevalent subtypes of HIV-1 in serodiscordant couples. Setting: Integrated Counselling and Testing Centre (ICTC), Department of Microbiology. Study Design: Prospective pilot study. Participants: Thirty HIV-1 serodiscordant couples. Inclusion Criteria: a) Documentation of HIV-1 infection in one partner and seronegative status in the other, current history of continued unprotected sexual activity within the partnership, demonstration that they have been in a partnership for at least 1 year and are not currently on highly active antiretroviral therapy HAART; b) willingness of both partners to provide written informed consent including consent to continued couple counselling for 3 months. Materials and Methods: HIV-1 subtyping was carried out by heteroduplex mobility analysis (HMA) by amplifying env region; and DNA sequencing by amplifying gag region. Results: HIV-1 env gene was amplified successfully in 10/30 samples; gag gene, in 25/30 samples; and both env and gag gene were amplified successfully in 5/30 samples. HIV-1 subtype C was detected from 21 samples; subtype B, from 7; and subtype A, from 2. Sample from 1 positive partner was detected as subtype C by env HMA and subtype B by gag sequencing. Conclusion: HIV-1 subtype C was found to be the predominant subtype of HIV-1 in serodiscordant couples attending our ICTC, followed by HIV-1 subtype B and HIV-1 subtype A, respectively. DNA sequencing was found to be the most reliable method for determining the subtypes of HIV-1.

PCR-heterodúplex por agrupamiento: implementación de un método de identificación de portadores de la mutación más común causal de fibrosis quística en Colombia / PCR-heteroduplex by grouping: rapid screening carrier method for cystic fibrosis F508del mutation in Colombia

Introducción: La fibrosis quística (FQ) es una enfermedad autosómica recesiva frecuente, con una incidencia de 1 en 2,500 recién nacidos. La causan más de 1,300 mutaciones distintas en el gen regulador de la conductancia transmembranal de la fibrosis quística (CFTR). Sin embargo, la mutación F508del es la más común en la mayoría de las poblaciones. Objetivos: Desarrollo de una técnica rápida, de bajo costo y confiable que permita filtrar con rapidez a los portadores o afectados por esta mutación que mediante el asesoramiento genético, contribuya a disminuir la aparición de nuevos casos y a un diagnóstico temprano de los enfermos y así lograr un descenso en la morbilidad y la mortalidad asociadas con la fibrosis quística en Colombia. Metodología: En el presente estudio se aplicó la técnica PCR-heterodúplex por agrupamientos, gracias al análisis de 400 muestras de sangre en papel filtro obtenidas de individuos asintomáticos para la FQ. Resultados: En las pruebas de validación de la técnica PCR-heterodúplex por agrupamiento se obtuvo una eficiencia, reproducibilidad y especificidad de 100% y una sensibilidad de 92%. Conclusiones: Se demostró la sensibilidad y reproducibilidad de la técnica PCR Directa-heterodúplex por agrupamientos de hasta 10 muestras, que se pueden emplear en programas para filtrar heterocigotos y afectados de F508del.

Background: Cystic fibrosis (CF) is the most frequent autosomal recessive disorder in the Caucasian population with an incidence of 1 in 2,500 newborns. More than 1,300 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that causes CF have been described. However, mutation F508del is the most common mutation in different populations around the world. Objective: To develop a fast, reliable and low-cost technique to screen carriers and affected individuals for the F508del mutation. This kind of analysis will have an impact on genetic counselling to decrease the incidence of new cases, in the early diagnosis and instauration of appropriate treatment to decrease morbidity and mortality associated to CF in Colombia. Methods: The reliability of the PCR-heteroduplex by grouping technique by analysis of 400 blood spot samples from asymptomatic CF patients was defined. Results: Using PCR-heteroduplex by grouping technique 100% efficiency, reproducibility and specificity and 92%sensitivity were found.Conclusions: The sensitivity and reproducibility of the PCR-heteroduplex by grouping technique up to pooling of 10 samples were demonstrated. This kind of analysis could be used in heterozygotes and affected screening programs.

Quasispecies of HCV before and after Interferon Therapy and Its Dynamic Change Detected by HMA / 中华医院感染学杂志

OBJECTIVE To investigate the quasispecies diversity and the dynamics of quasispecies evolution in patients with chronic Hepatitis C virus(HCV) infection.METHODS Changes of HCV quasispecies before and after interferon(IFN) therapy were detected by heteroduplex mobility analysis(HMA) and by automatic sequencer.RESULTS We found that no changes in viral complexity were observed with therapy,but there were marked changes in viral divergence.The proportion of major pretreatment variants was different in HVR1 in all patients.During the treatment,HCV quasispecies changed significantly.CONCLUSIONS HMA is a simple,rapid and reliable method for detecting HCV quasispecies.Responsiveness to interferon may be related to the changes in viral divergence,but not related to the numbers of viral variants.

Polymerase Chain Reaction and Heteroduplex Analysis Based Detection of Clonal T Cell Receptor Gamma Gene Rearrangements in Paraffin-embedded Tissues of Cutaneous T Cell Proliferative Diseases

BACKGROUND: Recently, the molecular pathologic investigation for clonality in lymphomas has been introduced and has gained a role in the diagnosis of lymphomas. In fact, the clonality test using TCRGR phenomenon has been done by Southern blot analysis (SBA) and polymerase chain reaction (PCR) for molecular pathologic diagnosis of T cell lymphomas. However, it is difficult to perform SBA with paraffin embedded specimens or with samples of small skin biopsies. OBJECTIVE: We investigated the efficacy of PCR amplification of TCR gene in paraffin em-bedded cutaneous T cell lymphomas. METHODS: Iii this study, the clonality was assessed by polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR) gene from the DNA extracts obtained from paraffin em-bedded tissues (PET) of malignant T cells, B cell lymphomas, and benign cutaneous T cell proliferative disorders. Heteroduple-x-analyses were also performed to rule out the false positives. RESULTS: Among the total of 62 cases analyzed, monoclonality was observed in 4 out of 10 mycosis fungoides, 7 out of 9 cutaneous T cell lymphomas excluding mycosis fungoides, 1 out of 3 angiocentric lymphomas, 2 out of 2 lymphomatosis papulosis, 1 out of 7 large plaque parapsoriasis, and 1 out of 2 T cell lymphomas in other organs. No monoclonality was observed in 9 inflammatory cutaneous diseases, 5 small plaque parapsoriasis, 4 cutaneous B cell lymphomas, and 11 B cell lymphomas in lymph nodes. CONCLUSION: The results suggest that the PCR method and heteroduplex analysis used in this study were not only practical but also efficacious for the diagnosis of cutaneous T cell lymphomas using tissues embedded in paraffins.

Molecular Diagnosis of Cutaneous T Cell Lymphoproliferative Diseases

It is often problematic to diagnose T-cell lymphoproliferative disorders of the skin because of the difficulty in establishing clonality in paraffin-embedded tissue. We used polymerase chain reaction single strand conformational polymorphism (PCR-SSCP) and heteroduplex analysis in paraffin embedded tissue to detect clonal rearrangement of T-cell receptor gamma (TCRgamma) gene in 17 T-cell lymphoproliferative disorders and 6 atypical lymphoproliferative diseases. We used polymerase chain reaction to detect TCR beta gene rearrangement in 8 of 17 cases which did not show TCRgamma gene rearrangement. Jurkat cell lines were used as monoclonal controls. DNA was extracted from 5 biopsies of T-cell lymphomas, 10 biopsies of mycosis fungoides, 2 biopsies of lymphomatoid papulosis, and 6 biopsies of atypical lymphoproliferative lesions. We detected monoclonality in 5 of 5 T-cell lymphoma cases, 2 of 2 lymphomatoid papulosis cases, 6 of 10 mycosis fungoides cases, and 2 of 6 atypical lymphoproliferative disease cases. We conclude that nonradioactive PCR-SSCP for TCR gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of cutaneous T cell lymphoproliferative disorders in paraffin embedded tissue.

Identification of two novel mutations of KEL alleles in Chinese population / 中国输血杂志

A mutation of intron 7, the point mutation in exon 9 was synonymous. Conclusion Two novel mutations of KEL gene are identified.

Association betwen Mannose-binding lectin structural gene polymorphisms and susceptibility of SLE in Han Nationality population from Hubei province of China / 中国免疫学杂志

0.05).Conclusion:MBL B allele is not a risk component in the developing process of SLE Chinese patients.