Touchdown PCR Research Articles

Identification and validation of a new male sex-specific ISSR marker in pointed gourd (Trichosanthes dioica Roxb.).

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

Polymorphisms in SNP CGIL4: association stuty on the phenotype of clinical mastitis resistance in Holstein cows

Bovine mastitis is the most important udder pathology and have a significant economic impact in the dairy industry. Its etiology is associated to problems with health and milking management. However, there are animals with resistance or susceptibility to mastitis, even when environmental factors are controlled. Recently, some molecular markers were associated to the phenotype of resistance to mastitis. The present study aims to verify if, based on clinical history of cows of second and third lactation, the SNP CGIL4 is associated to the phenotype of resistance to mastitis in Holstein cows in a herd in southern Brazil. Genomic DNA was obtained from blood samples of 160 Holstein cows (second and third lactation) from dairy herds from Rio Grande do Sul state, Brazil. Phenotype of resistance or susceptibility to clinical mastitis was defined based on animal’s clinical history, and the animal was classified as susceptible if presented at least 3 episodes of clinical mastitis during the last lactation. The identification of the CGIL4 SNP was performed using a PCR-RFLP. The association between genotipes and phenotipes was acessed by the χ 2 test. To detect polymorphism, Polymerase Chain Reaction (PCR) was performed using a touch-down PCR reaction producing an amplicon of 399 bp. The Restriction Fragment Length

Modification of PCR Conditions and Design of Exon-Specific Primers for the Efficient Molecular Diagnosis of PKD1 Mutations

Background/Aims: Autosomal-dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by mutations in the PKD1 and PKD2 genes. Currently, long-range PCR followed by nested PCR and sequencing (LRNS) is the gold standard approach for PKD1 testing. However, LRNS is complicated by the high structural and sequence complexity of PKD1, which makes the procedure for amplification and analysis of PKD1 difficult. Methods: Here in, we modified the PCR conditions and designed primers for efficient and specific amplification of both the long-range and individual exons of PKD1. Results: Using the modified system, seven long-range fragments were specifically amplified using two distinct sets of conditions, and all individual exon PCR assays were easily performed using a touch-down PCR method. Seven pathogenic or likely pathogenic variants, including two novel truncated frameshift indels and two novel likely pathogenic missense mutations, were identified in eight unrelated patients with or without histories of ADPKD disease (one variant was observed in two unrelated patients). Using combined bioinformatics tools, two indeterminate missense variants were identified in two sporadic patients. Conclusion: Four novel PKD1 variants were identified in this study. We demonstrated that the modified LRNS method achieves high sensitivity and specificity for detecting pathogenic variants of ADPKD.

Transposon Insertion Phenomenon during Cloning of a Partial Fragment Derived from Metagenomic DNA Isolated from Deep-Sea Water and Sediment of Kawio Island, North Sulawesi

Transposon is well-known as mobile element found abundant both in prokaryote and eukaryote genomes. In bacteria, transposon (famous name of a transposable DNA) could jump from chromosome to plasmid and its contrary. One type of transposons in bacteria known as insertion sequence (IS), it does not contain any additional genes except a gene encoding transposase, an enzyme that correlated to transponsition activities. The finding of transposon insertion unfortunately found during cloning of a fragment derived from deep-sea metagenomic DNA in this research. In the initial, this research was aimed to clone and characterize the á-amylase encoded gene derived from metagenomic DNA isolated from deep-sea water and sediment of Kawio Island, North Sulawesi. Metagenomic DNA has been isolated from deep-sea water and sediment and by using Whole Genome Amplification (WGA) technique, the DNA it could be increased in quantities to 146,31 ng for each 1 ng of metagenomic DNA. A fragment of ~1000 bp in length was obtained by using touchdown PCR method. The presence of a transposon in this DNA fragment is proposed as a hypothesis for losing ~700 bp leaving just 310 bp cloned sequence. Analysis of sequencing result showed a highest similarity between this 310 bp partial fragment with a replication protein (Rep) encoded gene from Pseudomonas putida (Query Coverage: 88%; Max. Identity: 80%, Positive: 86%) and this protein is known to be involved in plasmid replication where transposase encoding genes known usually presence together with this gene (Rep gene) in a bacterial plasmid.

Tolerance of an aerobic denitrifier (Pseudomonas stutzeri) to high O2 concentrations

An aerobic denitrifier was isolated from activated sludge and the isolate possessed an average removal rate of 5.7 mg NO3 (-)-N l(-1) h(-1) without accumulation of NO2 (-)-N (less than 2.1 mg l(-1)). The average removal efficiency of nitrate was 93.7 % in 24 h, when the dissolved oxygen (DO) concentrations ranged from 3.2 to 17.5 mg l(-1). The activity of both nap (periplasmic nitrate reductase) and nir (nitrite reductase), whose corresponding genes (napA and nirS) were amplified by touchdown PCR, could be responsible for the tolerance of DO concentrations. Other three genes relating to narG, norB and nosZ were noted to involve in isolate strain.

Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii

An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation

The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre–loxP system and a removable G418r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop-out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling

A Generic, Flexible Protocol for Preimplantation Human Leukocyte Antigen Typing Alone or in Combination with a Monogenic Disease, for Rapid Case Work-up and Application

Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for β-thalassemia (β-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.

Detection of a novel intragenic rearrangement in the creatine transporter gene by next generation sequencing

Deficiency caused by mutations in the creatine transporter gene (SLC6A8/CT1) is an X-linked form of intellectual disability. The presence of highly homologous pseudogenes and high GC content of SLC6A8 genomic sequence complicates the molecular diagnosis of this disorder. To minimize the pseudogene interference, exons 2 to 13 of SLC6A8 were amplified as a single PCR product using gene-specific long-range PCR (LR-PCR) primers. The GC-rich exon 1 and its flanking intronic sequences were amplified separately in a short fragment under GC-rich conditions and a touchdown PCR program. Traditional Sanger sequence analysis of all coding exons of SLC6A8 from a 3-year-old boy with creatine transporter deficiency did not detect deleterious mutations. The long-range PCR product was used as template followed by massively parallel sequencing (MPS) on HiSeq2000. We were able to detect a tandem duplication involving part of exons 11 and 12 in the SLC6A8 gene. The deduced c.1592_1639dup133 mutation was confirmed to be a hemizygous insertion by targeted genomic DNA and cDNA Sanger sequencing. Combination of deep sequencing technology with long-range PCR revealed a novel intragenic duplication in the SLC6A8 gene, providing a definitive molecular diagnosis of creatine transporter deficiency in a male patient.

Utilization of a Touchdown PCR Strategy to Amplify Low Coverage Regions and Variants Defined by Next Generation Sequencing

Next-generation sequencing (NGS) provides a cost-effective means for simultaneously interrogating vast amounts of the genome. Even with target enrichment strategies, some regions of the genome remain difficult to capture. We utilized a custom-designed SureSelect (Agilent, Santa Clara, CA) target enrichment followed by sequencing using a HiSeq 2000 (Illumina, San Diego, CA) to sequence the coding regions of 568 clinically …

Isolation and characterization of 23 novel polymorphic microsatellite markers from the endangered Puerto Rican boa (Chilabothrus inornatus) using paired-end Illumina shotgun sequencing

We developed 23 microsatellite loci for the endangered Puerto Rican boa, Chilabothrus inornatus, from 100 bp reads obtained from a paired-end Illumina shotgun library. Importantly, these loci can be amplified using the same touchdown PCR program. All loci were highly polymorphic, with between 5 and 15 alleles found among 24 individuals genotyped from three populations. We anticipate that these loci will be extremely useful in conservation genetic studies of this species.

A case report on Kennedy' s disease diagnosed by dynamic mutation of androgen receptor gene and mitochondrial gene analysis

Objective To diagnose a patient Kennedy' s disease,who was considered as mitochondrial myopathy by dynamic mutation of androgen receptor gene (AR) and mitochondrial gene analysis.Methods Dynamic mutation of AR gene was analyzed by PCR and sequencing,and mtDNA gene was analyzed by touch down PCR and sequencing.Results trinucleotide repeats of CAG were up to 46,and no pathological mutation of mtDNA was found.Conclusion Dynamic mutation analysis of AR gene and mitochondrial gene analysis are indispensable in diagnosing Kennedy' s disease. Key words: Kennedy' s androgen receptor gene (AR); Dynamic mutation; Mitochondrial gene analysis

Identification of human bocaviruses in the cerebrospinal fluid of children hospitalized with encephalitis in China

BackgroundEncephalitis is a major cause of death and disability in adults and children; different members in the family Parvoviridae are known to be associated with encephalitis to some extent. ObjectivesTo determine the presence of human bocaviruses (HBoVs) and corresponding HBoV-specific immunoglobulins (Igs) in cerebrospinal fluid from children with suspected viral encephalitis. Study designEmploying real-time PCR and nested touchdown PCR, 67 cerebrospinal fluid (CSF) samples from children with suspected viral encephalitis were screened for HBoV and routine encephalitis-causing viruses. Using ELISA, Western blot and IFA, HBoV-specific Ig were determined in the samples. ResultsNine samples (134%) were HBoV1 DNA-positive, while one sample (15%) was HBoV2 DNA-positive. HBoV-specific IgM and IgG antibodies were detected in the CSF samples from three children; two samples were HBoV1 DNA-positive and one sample was negative. One death was recorded; CSF from this child was the only HBoV-IgM-positive CSF samples among the 67 CSF tested. ConclusionWe screened CSF samples obtained from children with encephalitis for the presence of HBoV1 and HBoV2 and specific IgM- or IgG-responses. Detection of viral DNA and/or immunological response to HBoV1/HBoV2 highlights the significance of these viruses as causes of encephalitis in children. Further studies are needed to examine the role of HBoV infection in children encephalitis.

Isolation an Aldehyde Dehydrogenase Gene from Metagenomics Based on Semi-nest Touch-Down PCR

Culture-independent approaches to analyze metagenome are practical choices for rapid exploring useful genes. The mg-MSDH gene, acquired from the hot spring metagenomic, was retrieved full lengths of functional gene using semi-nest touch-down PCR. Two pairs of degenerate primers were used to separate seven conserve partial sequences by semi-nest touch-down PCR. One of them showed similarity with aldehyde dehydrogenase was used as a target fragment for isolating full-length sequence. The full-length mg-MSDH sequence contained a 1,473bp coding sequence encoding a 490-amino-acid polypeptide and assigned an accession number JQ715422 in Genbank. The upstream sequences TAGGAG of the start codon (GTG), suggested that was a ribosome binding site. The coding sequence of mg-MSDH was ligated to pET-303 vector and the reconstructive plasmid was successfully overexpressed in E. coli. The purified recombinant mg-MSDH enzyme showed propionaldehyde oxidative activity of 3.0 U mg(-1) at 37°C.

Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome

Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence.

β-Glucan Synthase Gene Overexpression and β-Glucans Overproduction in Pleurotus ostreatus Using Promoter Swapping

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

Overexpression, purification, molecular characterization and pharmacological evaluation for anticancer activity of ribosomal protein S23 from the giant panda (Ailuropoda melanoleuca)

Ribosomal protein S23 (RPS23) is a component of the 40S small ribosomal subunit encoded by the RPS23 gene, which is specific to eukaryotes. The cDNA and genomic sequence of RPS23 were cloned from Ailuropoda melanoleuca (A. melanoleuca) using reverse transcription‑polymerase chain reaction (RT-PCR) technology and touchdown PCR, respectively. The two sequences were analyzed preliminarily and the cDNA of the RPS23 gene was overexpressed in Escherichia coli (E. coli) BL21. The cDNA of RPS23 cloned from giant panda was 472 bp, and it contained an open reading frame (ORF) of 432 bp encoding 142 amino acids. The nucleotide sequence of the coding sequence showed a high degree of homology to some mammals as determined by BLAST analysis, similar to the amino acid sequence. The genomic sequence was 2,105 bp in length, with 4 exons and 3 introns. The primary structure analysis revealed that the molecular weight of the putative RPS23 protein was 15.80 kDa with a theoretical isoelectric point (pI) of 11.23. The molecular weight of the recombinant protein RPS23 was 21.5 kDa with a theoretical pI of 10.57. Topology prediction showed that there are seven different patterns of functional sites in the RPS23 protein of giant panda. RPS23 was successfully expressed in E. coli and its protein fused with the N‑terminal His‑tagged protein triggered the accumulation of an expected 21.5‑kDa polypeptide. The inhibitory rate of tumor growth in mice treated with 0.1 µg/ml RPS23 protein was 49.45%, the highest in the three doses used, which may be comparable to mannatide treatment. Histology of immune organs showed that the tissues were characterized by a regular and tight arrangement, while tumor tissues of the mice in the RPS23 group exhibited a loose arrangement compared to the control group. However, there was no obvious damage to other organs, such as the heart, lung and kidney. Investigations are currently being conducted to determine the bioactive principles of the recombinant protein RPS23 responsible for its anticancer activity.

CAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants

The availability of a simple, reproducible and cost-effective molecular marker is a prerequisite for plant genetic analysis. We have developed a novel promoter-targeted marker, CAAT box- derived polymorphism (CBDP) using the nucleotide sequence of CAAT box of plant promoters. CBDP, like random amplified polymorphic DNA (RAPD), uses single primer in polymerase chain reaction (PCR) for generating markers. However unlike RAPD, the CBDP primers are 18 nucleotides long and consist of a central CCAAT nucleotides core flanked by the filler sequence towards the 5′ end and di- or trinucleotides towards the 3′ end. In this study, a small set of 25 CBDP primer was designed and initially tested in a representative set of eight cultivars of jute for generation of polymorphic markers. Further, to achieve high reproducibility, a touchdown PCR was employed with an annealing temperature of 50oC. All the CBDP primers generated polymorphic markers in jute cultivars, and an UPGMA dendrogram based on Jaccard’s similarity grouped them into two clusters represented by Corchorus capsularis and C. olitorius, respectively. Interestingly, such grouping of jute cultivars was consistent with genetic relationships established earlier for these cultivars using other DNA markers. Moreover, these CBDP primers also generated polymorphic markers in representative sets of cotton (Gossypium species) and linseed (Linum usitatissimum ) cultivars. Given the high success rate of CBDP primers in generating markers in the tested species and advantages like ease in marker development and assay with reproducible profiles, they could potentially be exploited in other species as well for assessing genetic diversity, cultivar identification, construction of linkage map and marker- assisted selection.

Analyzing Arthropods for the Presence of Bacteria

Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis.

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 102 CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.