Primers For Polymerase Chain Reaction Amplification Research Articles

POSoligo software for in vitro gene synthesis

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5′- and 3′-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

Validation of Circular RNAs by PCR.

High-throughput RNA-sequencing (RNA-seq) technologies combined with novel bioinformatic algorithms discovered a large class of covalently closed single-stranded RNA molecules called circular RNAs (circRNAs ). Although RNA-seq has identified more than a million circRNAs, only a handful of them is validated with other techniques, including northern blotting, gel-trap electrophoresis, exonuclease treatment assays, and polymerase chain reaction (PCR). Reverse transcription (RT) of total RNA followed by PCR amplification is the most widely used technique for validating circRNAs identified in RNA-seq. RT-PCR is a highly reproducible, sensitive, and quantitative method for the detection and quantitation of circRNAs. This chapter details the basic guidelines for designing suitable primers for PCR amplification and validation of circRNAs .

Evaluation of the diagnostic value of immunoglobulin clonal gene rearrangements in patients with parotid gland MALT lymphoma using BIOMED-2 protocol

The aim of this study was to evaluate the diagnostic value of immunoglobulin (Ig) clonal gene rearrangements for mucosa-associated lymphoid tissue (MALT) lymphoma of the parotid gland. We collected and retrospectively analyzed clinical data of 21 patients referred to our institution between 2009 and 2017. Eight patients had been primarily diagnosed MALT lymphoma of the parotid gland and the remaining patients with lymphoepithelial lesion. Paraffin-embedded tissues were chosen for extracting genomic DNA and multiplex primer polymerase chain reaction amplification by using BIOMED-2 primers. Polymerase chain reaction amplification products were analyzed by heteroduplex analysis. Generally, 17 patients were identified to have parotid gland MALT lymphoma; 47.06% of them had Sjögren syndrome. The sensitivity of IGH VH-JH FR1, FR2, FR3, IGK Vκ-Jκ, and IGK (Vκ-Kde and intron-Kde) as targets was 76.47%, 82.35%, 88.24%, 29.41%, and 35.29%, respectively. The sensitivity of combined application of the above-mentioned 3 IGH primers as targets was 100%. The sensitivity of combined application of the above two IGK primers as targets was 58.82%. Ig clonal gene rearrangements assays using BIOMED-2 protocol can be a highly reliable diagnostic method for parotid gland MALT lymphoma. For patients with Sjögren syndrome along with histologically benign lymphoepithelial lesion, identification of Ig clonal gene rearrangements is important for routine differential diagnosis.

Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study.

AimTo determine polymerase chain reaction (PCR)-based blood group on tooth pulp obtained from teeth stored for 1 month, 6 months, and 1 year following extraction and to evaluate the stability of deoxyribonucleic acid (DNA) in primary tooth subjected to a temperature of 200°C ± 5°C for 15 minutes.Materials and methodsDental pulp tissue was collected from 40 exfoliated primary teeth stored for various time durations and temperature and preserved at 4°C till DNA extraction was carried out. Deoxyribonucleic acid was extracted using silica membrane-based spin-column procedure of QIAamp DNA minikit from BioRad. Deoxyribonucleic acid was subjected to PCR amplification and monoplex allele-specific PCR primers for ABO genotyping.Statistical analysis usedThe data were analyzed by comparison (based on percentage).ResultsIn our study, overall, 85% samples showed a DNA yield. Cent percent results were obtained for samples studied at the end of 1 month followed by 90 and 80% for samples studied for 6 months and 1 year respectively. Heated samples showed 70% result.ConclusionPolymerase chain reaction was found to be an effective method for blood group determination for teeth stored at various time durations and temperatures. However, as the time interval increased, the number of positive results obtained decreased.How to cite this articlePai RK, Bhat SS, Salman A, Hegde S. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study. Int J Clin Pediatr Dent 2016;9(4):308-312.

Human papillomavirus type 197 is commonly present in skin tumors

Non-melanoma skin cancers commonly contain Human Papillomavirus (HPV), but the types found have varied depending on the polymerase chain reaction (PCR) primer systems used. Whole genome amplified DNA (not amplified by any specific PCR primers) from 91 skin lesions [41 squamous cell skin carcinomas (SCCs), 8 keratoacanthomas, 22 actinic keratoses, 3 basal cell carcinomas and 17 SCCs in situ] were sequenced. All samples were sequenced both at 160 Mb and 1.8 Gb sequencing depth per sample. The sequences from 10 different HPVs in 47/91 specimens were found. Sequences represented four established HPV types (HPV types 16, 22, 120, 124), two previously known putative types (present in GenBank) and four previously unknown HPV sequences (new putative types). The most commonly detected virus was cloned, sequenced and designated as HPV197. Type-specific real-time PCR detected HPV197 in 34/91 specimens. For comparison, a pool of the same samples after general primer PCR amplification was also sequenced. This revealed 40 different HPVs, but only two HPV types were detected both with sequencing without prior PCR and with sequencing PCR amplicons, suggesting that sequencing without prior PCR gives a more unbiased representation of the HPVs present. In summary, it was found that HPV can be sequenced from most skin disease specimens and HPV197 appeared to be the most commonly present virus.

Identification of the Bacterial Community Responsible for Decontaminating Eleme Petrochemical Industrial Effluent Using 16s RDNA PCR Denaturing Gradient Gel Electrophoresis

Identification of bacteria community responsible for decontaminating Eleme petrochemical industrial effluent using 16S PCR denaturing gradient gel electrophoresis (DGGE) was determined. Gene profiles were determined by extracting DNA from bacterial isolates and amplified by polymerase chain reaction (PCR) using 16S rDNA eubacterial primers with a GC clamp. PCR amplified DNA was separated by Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Comparative evaluation of primers for PCR amplification of DNA revealed that V6V8F and V6V8R primers were most suitable for amplification DNA. Denaturing gradient gel ratio of 20% : 80% was most suitable for differentiation of bacterial profiles. Sequencing and phylogenetic analysis of DGGE products were successful displaying 88% -100% similarity and homology to Roseococcus sp. LW5(FM956480.1). Dendograms and similarity matrix revealed 81.5% -100% from sampling points.http://dx.doi.org/10.4314/njt.v33i4.6

Melting Profiles May Affect Detection of Residual HPV L1 Gene DNA Fragments in Gardasil<sup>®</sup>

Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

Analysis of the NMI01 Marker for a Population Database of Cannabis Seeds

We have analyzed the distribution of genotypes at a single hexanucleotide short tandem repeat (STR) locus in a Cannabis sativa seed database along with seed-packaging information. This STR locus is defined by the polymerase chain reaction amplification primers CS1F and CS1R and is referred to as NMI01 (for National Marijuana Initiative) in our study. The population database consists of seed seizures of two categories: seed samples from labeled and unlabeled packages regarding seed bank source. Of a population database of 93 processed seeds including 12 labeled Cannabis varieties, the observed genotypes generated from single seeds exhibited between one and three peaks (potentially six alleles if in homozygous state). The total number of observed genotypes was 54 making this marker highly specific and highly individualizing even among seeds of common lineage. Cluster analysis associated many but not all of the handwritten labeled seed varieties tested to date as well as the National Park seizure to our known reference database containing Mr. Nice Seedbank and Sensi Seeds commercially packaged reference samples.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Diptera-Specific Polymerase Chain Reaction Amplification Primers of use in Molecular Phylogenetic Research

Abstract DNA sequence data from a variety of mitochondrial and nuclear gene regions are significant components of phylogenetic research in entomology. Polymerase chain reaction (PCR) amplification primers for many gene regions have been developed that are specific to a range of dipteran groups. Here, we review the existing Diptera-specific PCR amplification primers that have been published for 11 mitochondrial and nuclear gene regions: 12S small ribosomal subunit, cytochrome b, cytochrome oxidase c subunit I, 28S ribosomal RNA, alanyl-tRNA synthetase, the carbamoyl phosphate synthase region of CAD, elongation factor-1α, 6-phosphogluconate dehydrogenase, triose phosphate isomerase, white, and wingless. We also have designed in total 94 new PCR amplification primers for use in these same gene regions. Our new primers have been developed and tested using our DNA sequence database of > 1,600 specimens representing 40 families of Diptera. All of the past and newly developed primer sequences are presented in tables, and their locations are shown on gene maps. This combined data will facilitate future molecular phylogenetic research within Diptera.

Disruption of a Ciliary B9 Protein Complex Causes Meckel Syndrome

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

Detection of cutaneous HPV types 4 and 24 DNA sequences in breast carcinoma in Singaporean women of Asian ancestry

To date, there have been conflicting data on the prevalence of HPV in primary breast carcinoma, with a median prevalence of 35%. We believe that the low prevalence reported could be due to use of inappropriate amplification primers and methods of detection. We designed a study to detect and reflect more accurately the incidence of both mucosal and cutaneous HPV types in breast carcinoma among Singaporean women. In our study, we used two different molecular techniques, both of which involved a polymerase chain reaction (PCR) amplification step for any HPV DNA found in the breast cancer tissues. The first method targeted primarily the mucosal HPV types, and the second, primarily the cutaneous HPV types. Formalin-fixed, paraffin-embedded breast cancers were used for the studies. The first method involved the GP5+/GP6+ primers for PCR amplification and a commercially available HPV DNA genotyping chip for detection. The subsequent studies involved the use of the FAB 59/64 primers for amplification, followed by DNA sequencing and comparison to the NCBI GenBank database for the detection of all possible HPV types. With the first technique, all 92 breast cancers tested gave negative results for HPV DNA, suggesting the absence of HPV types in breast cancers. Using the second method, we detected HPV sequences in 32/92 (35%) samples, of which 28 were shown to be HPV-4, one was HPV-24, two had mixed HPV types and one had an indeterminate HPV sequence that did not match any of the HPV sequences deposited in the GenBank database. These results were consistent with our hypothesis that the true incidence of HPV in breast carcinoma is much higher than those reported to date, and that this is probably due to the limited sensitivity of the molecular techniques used in earlier studies.

Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.

Analysis of oncogenic AKT1 p.E17K mutation in carcinomas of the biliary tract and liver

Sir, We have read with great interest the article by Kim et al (2008), in which they report the analysis of 713 cancer tissues for somatic mutations in the AKT1, AKT2 and AKT3 genes. They detected the previously reported AKT1 pleckstrin homology domain mutation (AKT1 p.E17K) in 4 out of 93 (4.3%) breast carcinomas, but not in the colorectal, lung, gastric and hepatocellular carcinomas of their series (Carpten et al, 2007). The authors concluded that the AKT1 p.E17K mutation should be further analysed in a wider range of cancers. We have studied 118 carcinomas of the biliary tract and liver (11 intrahepatic and 34 extrahepatic cholangiocarcinomas, 23 gallbladder carcinomas and 50 hepatocellular carcinomas) for AKT1 p.E17K mutations using polymerase chain reaction (PCR) and direct DNA sequencing. DNA was extracted from formalin-fixed, paraffin-embedded tumour tissues. The primers for PCR amplification and sequencing of exon 4 had the following sequence: forward 5′-CTGGCCCTAAGAAACAGCTCC-3′ and reverse 5′-CGCCACAGAGAAGTTGTTGA-3′. Reaction conditions for PCR amplification were 40 cycles of 95°C for 30 s, 60°C for 1 min and 72°C for 1 min. Polymerase chain reaction products were purified (GFX PCR DNA and Gel Band Purification Kit, Amersham Biosciences, Otelfingen, Switzerland) and analysed on an automated DNA sequencer (model 3130) using Gene Scan and SeqScape software (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were compared with the genomic sequence of AKT1 (ENS0142208). We did not observe AKT1 p.E17K mutations in any of the 118 carcinomas of the biliary tract and liver in our series. We have recently shown that the frequent activation of the PI3K/AKT pathway in carcinomas of the biliary tract and liver is associated with PIK3CA hot spot mutations in a small subset of these tumours (Riener et al, 2008). The low frequency of these mutations lead to the conclusion that further genetic changes have to be responsible for the activation of the PI3K/AKT pathway in these cancers. The recently identified AKT1 p.E17K mutation appeared to be a good candidate for such a mechanism. Our results in hepatocellular carcinomas confirm the findings of Kim et al in a different ethnic background and suggest that cholangiocarcinoma and gallbladder carcinoma can be added to the list of cancers that lack AKT1 p.E17K mutations.

New HLA-A, -B, and -C locus-specific primers for PCR amplification from cDNA: application in clinical immunology

The individual cellular immune response to intracellular antigens is modeled by the highly polymorphic major histocompatibility complex (HLA) class I molecules. The epitopes presented and the T cell repertoire that recognizes them depend on the HLA constitution of the individual. Therefore, to monitor and to modify an individual's HLA class I-driven cellular immune response, it is necessary to know the HLA class I alleles of the person and the possible epitopes of the target antigen presented by those alleles. In particular, this is necessary in order to design peptide-based vaccines and immune therapies for the treatment of diseases caused by viruses, intracellular parasites or cancer, and to monitor the immune response during those treatments. We describe a new set of HLA-A, -B, and -C locus-specific primers for the polymerase chain reaction (PCR) amplification of the whole coding sequence of these genes from complementary DNA (cDNA). We describe their use for typing and for the production of a library of recombinant HLA class I genes. We discuss two downstream applications of this gene collection: production of soluble HLA molecules and discovery of new epitopes.

Real-time sequence-specific primer polymerase chain reaction amplification of HLA class II alleles: a novel approach to analyze microchimerism.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

Circular viral DNA and anomalous junction sequence in PBMC of HIV-infected individuals with no detectable plasma HIV RNA.

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels <50 copies/ml of blood had detectable 2-LTR DNA. Sequencing of the products identified normal LTR--LTR junctions in the minority of cases with the majority containing anomalies including deletions and insertions. The persistence of HIV ccDNA in patients with no detectable plasma RNA could be consistent with ongoing de novo infection of dividing cells or with stability of this form of DNA in nondividing cells.

Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain.

A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital. Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea. Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1). Relapses occurred in three patients. Nineteen of the 27 episodes required anti-Clostridium difficile treatment. Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical. The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied. No phenotypic activity of enterotoxin A was detected. A deletion of approximately 1.7 kb was found in the toxin A gene. Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin.

Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.

Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects.

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.