Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.     

Simplified DNA Extraction and Improved PCR Based Detection of Greening Bacterium in Citrus

Citrus greening disease caused by a fastidious bacterium is an important graft transmissible disease in commercial citrus in India and other parts of the world. Polymerase chain reaction (PCR) is a sensitive and convenient method for detection of greening bacterium. A non-phenol chloroform method of DNA extraction was evaluated for DNA quality and PCR based detection of greening bacterium. The method was comparable with a commercial DNA extraction kit (Qiagen) and better than a CTAB based DNA extraction method. To improve the reliability, three primer sets (primers A, B, and C yielding amplicons of 1160 bp, 703 bp and 451 bp, respectively) and two polymerase enzymes (Taq polymerase and Klen Taq polymerase) were evaluated. The primer set C provided better amplification when compared to primer sets A and B. Primer C in combination with Taq polymerase provided amplification band at a DNA template concentration of 100 pg but good amplification band was obtained at still lower DNA template concentration of 0.1 pg when Klen Taq polymerase was used. The standardized PCR protocol combining non-phenol chloroform method of DNA isolation, primer set C and Klen Taq polymerase enzyme was found very effective in detecting greening bacterium in citrus trees. The sequence of cloned amplicon from 16S ribosomal RNA gene had 89–100 % sequence identity with corresponding sequence of Candidatus Liberibacter asiaticus from China, Brazil, Japan and Pune isolate of India, C. Liberibacter americnus from Brazil and C. Liberibacter africanus from Africa.     

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.1 pg of starting DNA template. Background cross-amplification with DNA templates derived from sixteen other species was negligible prior to 30 cycles of PCR. The species-specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect known quantities of species-specific DNA from mixed (complex) sources. The 10 assays reported here will help facilitate the sensitive detection and quantitation of common domestic animal and bird species DNA from complex biomaterials.     

Sequencing PCR DNA amplified directly from a bacterial colony

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.     

Assessment of amplicons in the DNA from boiled tissue by PCR and AP-PCR amplification

Polymerase chain reaction (PCR) is a technique sensitive enough to amplify small DNA fragments a billion-fold. The generation of amplicons either by PCR with a set of oligo primers or by arbitrarily primed AP-PCR with a single oligonucleotide primer is based on the availability of intact template and priming sites. With these approaches, it is possible to generate specific and random amplicons to assess the extent of damage to DNA caused by any of the physical, chemical, or environmental factors. We report the amplification of sex chromosome and autosome specific loci in the buffalo (Bubalus bubalis) genome by symmetrical and AP-PCR performed on DNA samples isolated from the muscle tissues that were boiled (treated) for different lengths of time. No difference was noticed in the amplification profile of DNA cooked for various lengths 0f time. However, after HinfI treatment, AP-PCR amplification of these DNAs revealed more bands on agarose gel than unrestricted samples. The successful amplification of the DNA samples isolated from the boiled tissues is attributed to the intactness of the amplicons. This suggests that despite storage for more than a year and subsequent heat treatment to the muscle tissues, the DNA remains a good substrate for PCR and AP-PCR amplification. Relevance of this work in the context of DNA probe technology is discussed.     

Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA

The use of formalin-fixed paraffin-embedded (FFPE) tissues for HPV DNA detection by PCR from biopsy materials is not entirely clear in retrospective studies. The aim of our study was to evaluate the usefulness and efficiency of FFPE tissues from laryngeal cancer (LSCC) in HPV detection by immunohistochemistry reaction (IHC) and PCR-DNA enzyme immunoassay method (PCR/DEIA) and to compare with HPV detection from DFT. HPV-DNA was amplified from 54 FFPE tissues from LSCC specimens by the short PCR fragment (SPF10) primer set using PCR/DNA method and monoclonal anti Human Papillomavirus antibodies in IHC. In the same patients 54 specimens were collected and immediately deep-frozen and stored at (-70°C) to (-80°C). All the FFPE and deep-frozen tissue (DFT) specimens were positive for β-globin amplification. HPV was detected by two methods (SPF10 PCR/DEIA and IHC) in 14 (25.92%) out of 54 specimens from FFPE. Significant differences were found between the HPV detection using PCR/DEIA method and IHC method in FFPE tissues. The comparative analysis of the 54 samples after assuming PCR method in FFPE tissues showed accuracy of 92.6%, sensitivity of 90.5% and specificity of 93.9%. The FFPE tissues method has high sensitivity, specificity and accuracy when used to detect HPV DNA by PCR reaction and it is comparable to DFT results. DNA quality of FFPE samples is adequate and it can be used in HPV-DNA detection and in retrospective studies on LSCC.     

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.     

A generally applicable assay for the quantification of inhibitory effects on PCR

PCR amplification of target genes from environmental DNA extracts can suffer from PCR inhibition, caused by co-extracted substances. No simple assay has been available to quantify this inhibition. Therefore, a generally applicable PCR inhibition-assay was developed, which allows determination of statistically significant inhibition of PCR. This information is important when documenting quality of DNA in environmental extracts used as template for PCR.     

Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.     

疱疹病毒定量PCR的建立

A single pair of oligonucleatide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competiitve PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method.The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of c…     

A Method to Evaluate Genome-Wide Methylation in Archival Formalin-Fixed,Paraffin-Embedded Ovarian Epithelial Cells

Background

The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.

Methodology/Principal Findings

Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.

Conclusions/Significance

We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.     

MPprimer: a program for reliable multiplex PCR primer design

Background  

Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility.     

Sources of PCR-induced distortions in high-throughput sequencing data sets

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

Whole Genome Amplification for Sequencing and Applications in Conservation Genetics

Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase-mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.     

Quantitative PCR in the diagnosis of Leishmania

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

丹参ISSR-PCR反应体系的建立与正交优化

利用正交试验设计的方法,从引物浓度、Taq DNA聚合酶浓度、Mg2+浓度、dNTP浓度4种因素3个水平,对丹参ISSR-PCR反应体系进行优化分析,并在此基础上对模板DNA浓度、PCR反应过程中的退火温度进行梯度检测。结果表明:20μL ISSR-PCR反应体系中各因素的最佳浓度为1×PCR buffer、200μmol/L dNTP、1.0μmol/L引物、1.5mmol/L Mg2+和1 U Taq DNA聚合酶,最佳模板DNA浓度为20～60ng,引物UBC 835的最佳退火温度为51.7℃。