Two-round Polymerase Chain Reaction Research Articles

Detection of Borrelia miyamotoi in Ixodidae Ticks Collected in the South of Western Siberia

Objective of the studywas to assess the level of infection with Borrelia miyamotoi in ticks Ixodes persulcatus and Ixodes pavlovskyi collected in the south of Western Siberia.Materials and methods.688 ticks I. persulcatus and I. pavlovskyi collected on the territory of Novosibirsk, Tomsk and Kemerovo Regions were examined. Borrelia DNA was detected by a two-round polymerase chain reaction with specifc primers to the omp66 gene region, followed by sequencing of the isolated fragments and phylogenetic analysis, including the sequences of prototype isolates published in the international GenBank database. Comparison and analysis of nucleotide sequences was carried out using the MEGA 7 software package. In parallel, the samples were examined for the presence of the agents of transmissible infections – West Nile fever and tick-borne encephalitis, anaplasmosis, ehrlichiosis, babesiosis, bartonellosis and candidate pathogens of tick-borne rickettsiosis through RT-PCR and PCR with species-specifc and genus-specifc primers.Results and discussion.The genetic material of B. miyamotoi was found in 2.2 % of I. persulcatus and I. pavlovskyi ticks in the Tomsk, Novosibirsk, and Kemerovo Regions and their appurtenance to the Asian genotype was determined. It was revealed that the degree of similarity of the omp66 gene fragments within the group of isolates of the Asian genotype was 100 %. No relation between B. miyamotoi and a specifc vector species was identifed.

Effect of antifouling dendrimers and Au DENPs on the enhancement of PCR amplification

The polymerase chain reaction (PCR) has been considered as one of the most fundamental techniques to amplify and analyze specific DNA fragments in the field of molecular biology and clinical medicine. Recently, a variety of nanoparticles (NPs) have been regarded as a novel method to enhance both the quality and yield of PCR technique. Herein, we report the use of generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers and dendrimer-entrapped gold nanoparticles (Au DENPs) modified with polyethylene glycol (PEG) moieties and (or) acetyl groups as a novel class of enhancers to improve the PCR amplification. We set up the nonspecific PCR and two-round PCR as model systems to investigate mechanisms of enhanced PCR. Our results show that dendrimer-based derivatives seem to enhance the PCR specificity. It is worth noting that the modification of antifouling PEG significantly lowered the optimization capability of the corresponding dendrimers, although this inhibition effect can be remarkably compromised by the entrapment of Au NPs. Furthermore, we found that in the presence of Au NPs, the thermal conductivity induced by Au NPs could play the dominant role in the PCR optimization, whereas in the absence of Au NPs, the electrostatic interaction of the dendrimers with PCR components may be the major factor affecting the PCR system. Our results also showed that the optimal concentrations of the materials in the two test systems were very close, which indicates that the developed dendrimer derivatives with good thermal stability may be the efficient PCR additives for enhancing different PCR systems.

Mechanistic Studies of Enhanced PCR Using PEGylated PEI-Entrapped Gold Nanoparticles.

The polymerase chain reaction (PCR) is considered an excellent technique and is widely used in both molecular biology research and various clinical applications. However, the presence of byproducts and low output are limitations generally associated with this technique. Recently, the use of nanoparticles (NPs) has been shown to be very effective at enhancing PCR. Although mechanisms underlying this process have been suggested, most of them are mainly based on PCR results under certain situations without abundant systematic experimental strategy. In order to overcome these challenges, we synthesized a series of polyethylene glycol (PEG)-modified polyethylenimine (PEI)-entrapped gold nanoparticles (PEG-Au PENPs), each having different gold contents. The role of the synthesized NPs in improving the PCR technique was then systematically evaluated using the error-prone two-round PCR and GC-rich PCR (74% GC content). Our results suggest a possible mechanism of PCR enhancement. In the error-prone two-round PCR system, the improvement of the specificity and efficiency of the technique using the PEG-Au PENPs mainly depends on surface-charge-mediated electrostatic interactions. In the GC-rich PCR system, thermal conduction may be the dominant factor. These important findings offer a breakthrough in understanding the mechanisms involved in improving PCR amplification, as well as in the application of nanomaterials in different fields, particularly in biology and medicine.

Deficient DNA mismatch repair is associated with favorable prognosis in Thai patients with sporadic colorectal cancer.

To determine the prognostic significance of deficient mismatch repair (dMMR) and BRAF V600E in Thai sporadic colorectal cancer (CRC) patients. We studied a total of 211 out of 405 specimens obtained from newly diagnosed CRC patients between October 1, 2006 and December 31, 2007 at Siriraj Hospital, Mahidol University. Formalin-fixed paraffin-embedded blocks of CRC tissue samples were analyzed for dMMR by detection of MMR protein expression loss by immunohistochemistry or microsatellite instability using polymerase chain reaction (PCR)-DHPLC. BRAF V600E mutational analysis was performed in DNA extracted from the same archival tissues by two-round allele-specific PCR and analyzed by high sensitivity DHPLC. Associations between patient characteristics, MMR and BRAF status with disease-free survival (DFS) and overall survival (OS) were determined by Kaplan-Meier survival plots and log-rank test together with Cox's proportional hazard regression. dMMR and BRAF V600E mutations were identified in 31 of 208 (14.9%) and 23 of 211 (10.9%) tumors, respectively. dMMR was more commonly found in patients with primary colon tumors rather than rectal cancer (20.4% vs 7.6%, P =0.01), but there was no difference in MMR status between the right-sided and left-sided colon tumors (20.8% vs 34.6%, P = 0.24). dMMR was associated with early-stage rather than metastatic disease (17.3% vs 0%, P = 0.015). No clinicopathological features such primary site or tumor differentiation were associated with the BRAF mutation. Six of 31 (19.3%) samples with dMMR carried the BRAF mutation, while 17 of 177 (9.6%) with proficient MMR (pMMR) harbored the mutation (P = 0.11). Notably, patients with dMMR tumors had significantly superior DFS (HR = 0.30, 95%CI: 0.15-0.77; P = 0.01) and OS (HR = 0.29, 95%CI: 0.10-0.84; P = 0.02) compared with patients with pMMR tumors. By contrast, the BRAF V600E mutation had no prognostic impact on DFS and OS. The prevalence of dMMR and BRAF V600E in Thai sporadic CRC patients was 15% and 11%, respectively. The dMMR phenotype was associated with a favorable outcome.

A highly effective polymerase chain reactionenhancer based on dendrimer-entrapped gold nanoparticles

In molecular biology, polymerase chain reaction (PCR) has played an important role but suffers a general problem of low efficiency and specificity. Development of suitable PCR additives to improve the specificity and efficiency still remains a great challenge. Here we report the use of dendrimer-entrapped gold nanoparticles (Au DENPs) as a novel class of enhancers to improve the specificity and efficiency of PCR. We show that the Au DENPs prepared using amine-terminated generation 5 poly(amidoamine) dendrimers (G5.NH(2)) as templates are much more effective than the same dendrimers without AuNPs entrapped in improving the specificity and efficiency of an error-prone two-round PCR system. With the increase of the molar ratio between Au atom and G5.NH(2) dendrimer in the Au DENPs, the optimum concentration of Au DENPs used to improve the PCR specificity and efficiency is decreased and can be as low as 0.37 nM when the Au atom/G5.NH(2) dendrimer molar ratio reaches 100:1. Our PCR results along with the dynamic light scattering data suggest that unlike the flexible soft dendrimers without NPs entrapped that may display a non-spherical shape when interacting with the PCR components, the Au DENPs with increasing Au atom/dendrimer molar ratio are able to reserve the spherical shape of dendrimers, enabling much more efficient interaction with the PCR components. Therefore, as a NP-based PCR enhancer, both the surface charge and the shape of the particles should be responsible for effective interaction with the PCR components for improving the PCR specificity and efficiency. Furthermore, the used Au DENPs were proved to be stable after the PCR process, enabling them to be potentially used for enhancing different PCR systems.

Effect of surface charge of polyethyleneimine-modified multiwalled carbon nanotubes on the improvement of polymerase chain reaction

In molecular biology, polymerase chain reaction (PCR) has played an important role but suffers a general problem with low efficiency and specificity. Development of suitable additives to improve the PCR specificity and efficiency and the understanding of the PCR enhancing mechanism still remain a great challenge. Here we report the use of polyethyleneimine (PEI)-modified multiwalled carbon nanotubes (MWCNTs) with different surface charge polarities as a novel class of enhancers to improve the specificity and efficiency of PCR. The materials used included the positively charged PEI-modified MWCNTs (CNT/PEI), the neutral CNT/PEI modified with acetic anhydride (CNT/PEI.Ac), and the negatively charged CNT/PEI modified with succinic anhydride (CNT/PEI.SAH). We show that the specificity and efficiency of an error-prone two-round PCR are greatly impacted by the surface charge polarity of the PEI-modified MWCNTs. Positively charged CNT/PEI could significantly enhance the specificity and efficiency of PCR with an optimum concentration as low as 0.39 mg L(-1), whereas neutral CNT/PEI.Ac had no such effect. Although negatively charged CNT/PEI.SAH could enhance the PCR, the optimum concentration required (630 mg L(-1)) was more than 3 orders of magnitude higher than that of positively charged CNT/PEI. The present study suggests that the PCR enhancing effect may be primarily based on the electrostatic interaction between the positively charged CNT/PEI and the negatively charged PCR components, rather than only on the thermal conductivity of MWCNTs.

Numerical Simulation of Au Nanoparticles Effect on the PCR Process

Gold nanoparticles have been found to greatly enhance the polymerase chain reaction (PCR) specificity and yield in recent studies. However, the underlying mechanism is still unclear, though different hypotheses have already been proposed. In this study, a mass-action based model has been developed to investigate the effect of Au nanoparticles on the two-round PCR results. The great affinity of Au nanoparticles to the single-stranded DNA is taken into consideration. Each nanoparticle is treated as a bioreactor and/or a selector, around which, reaction equations are coupled to simulate the particle effect, and to investigate the key parameters that might influence such an effect. It is assumed that there exists a competing mechanism between the specific and nonspecific bindings, both in the solution and on the particle surface during the reactions. The numerical predictions accord well to the experimental results, and can be used to explain the Au nanoparticles' effect on the enhancement of the PCR specificity and efficiency.

Enhanced specificity and efficiency of polymerase chain reactions using poly(amidoamine) dendrimers and derivatives

The polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Improvements to enhance the specificity and efficiency of PCR are always required and remain a great challenge. Here we introduce generation 4 (G4) and 5 (G5) poly(amidoamine) (PAMAM) dendrimers and their derivatives with different terminal groups as a novel class of enhancers to improve the specificity and efficiency of PCR. We show that the dendrimers are effective in optimizing error-prone two-round PCR and non-specific PCR in specificity and efficiency. With the increase of the number and density of terminal amine groups, the optimum concentration of polycationic dendrimers could be decreased to as low as 1.35 nM, which is four orders of magnitude smaller than that of fully acetylated dendrimers and carboxyl-terminated dendrimers. The present study underlines a fact that dendrimers could be used as a powerful additive to enhance the specificity and efficiency of PCR.

SIZE AND SURFACE EFFECT OF GOLD NANOPARTICLES (AuNPs) IN NANOGOLD-ASSISTED PCR

Recently, gold nanoparticles ( AuNPs ) were reported to increase the specificity and efficiency of the polymerase chain reaction (PCR). In this paper, we tested the enhancement of AuNPs with five different sizes on the specificity of two-round PCR. The results showed that, except 5.02 nm AuNPs , the AuNPs that could achieve the similar enhancement happened to have nearly the same total surface area. The surface effect seems to be the key factor of nanogold-assisted PCR.

Restriction Site-dependent PCR: An Efficient Technique for Fast Cloning of New Genes of Microorganisms

New bioactive proteins need to be screened from various microorganisms for the increasing need for industrial and pharmaceutical peptide, proteins, or enzymes. A novel polymerase chain reaction (PCR) method, restriction site-dependent PCR (RSD-PCR), was designed for rapid new genes cloning from genomic DNA. RSD-PCR strategy is based on these principles: (i) restriction sites disperse throughout genomes are candidacy for universal pairing; (ii) a universal primer is a combination of a 3′-end of selected restriction sites, and a 5′-end of degenerated sequence. A two-round PCR protocol was designed and optimized for the RSD-PCR: amplify the single strand target template from genomic DNA by a specific primer and amplify the target gene by using the specific primer and one of the universal RSD-primers. The optimized RSD-PCR was successfully applied in chromosome walking using specific internal primers, and cloning of new genes using degenerated primers derived from NH2-terminal amino acid sequence of protein.

Transfusion‐transmitted virus is not present in factor IX concentrates commonly used to treat haemophilia B

Transfusion-transmitted virus (TTV) is a potential cause of post-transfusion hepatitis in patients with haemophilia. Plasma-derived clotting factor concentrates currently undergo processes that are effective in removal and inactivation of viruses such as HIV, hepatitis B and C; however, their effectiveness with respect to TTV is unknown. To determine if TTV DNA is present in plasma-derived concentrates of factor IX, we tested 14 lots of Mononine and compared the results with BeneFix. Nucleic acid isolation, followed by a two-round polymerase chain reaction (PCR) and agarose gel analysis indicated that all 17 lots were negative for TTV. Although TTV may be considered an emerging pathogen, no evidence of the virus was detected in the commercially available plasma-derived concentrate of FIX most commonly used to treat haemophilia B.

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Direct polymerase chain reaction (PCR) detection of insertion/deletion (indel) polymorphisms requires sample homozygosity. For the indel polymorphisms that have the deletion allele with a relatively low frequency in the autosomal regions, direct PCR detection becomes difficult or impossible. The present study is, to our knowledge, the first designed to directly detect indel polymorphisms in a human autosomal region (i.e., the immunoglobulin V(H) region), through use of single haploid sperm cells as subjects. Unique marker sequences (n=32), spaced at approximately 5-kb intervals, were selected near the 3' end of the V(H) region. A two-round multiplex PCR protocol was used to amplify these sequences from single sperm samples from nine unrelated healthy donors. The parental haplotypes of the donors were determined by examining the presence or absence of these markers. Seven clustered markers in 6 of the 18 haplotypes were missing and likely represented a 35-40-kb indel polymorphism. The genotypes of the donors, with respect to this polymorphism, perfectly matched the expectation under Hardy-Weinberg equilibrium. Three V(H) gene segments, of which two are functional, are affected by this polymorphism. According to these results, >10% of individuals in the human population may not have these gene segments in their genome, and approximately 44% may have only one copy of these gene segments. The biological impact of this polymorphism would be very interesting to study. The approach used in the present study could be applied to understand the physical structure and diversity of all other autosomal regions.

Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over.

For the detection of hepatitis C virus (HCV) specific nucleic acids the polymerase chain reaction (PCR) is widely used. Rapid-cycle PCR is performed in glass capillaries with the LightCycler instrument and allows PCR including product analysis to be performed within a closed system in about 1 h. Thus, rapid-cycle PCR appears especially suitable for routine diagnostic applications. However, the volume of the PCR vessel is restricted to about 20 microl, which may limit the sensitivity of the PCR. To increase its sensitivity two-round or nested primer PCR protocols have been developed. In rapid-cycle PCR first-round PCR products are usually collected from the capillaries by centrifugation, a procedure prone to cross-contamination. Development of a two-round rapid-cycle reverse transcription-polymerase chain reaction (RT-PCR) in single closed LightCycler capillaries for the sensitive detection of HCV RNA in serum or plasma. A set of two pairs of nested primers was selected. The first-round RT-PCR reaction mixture was separated from the second-round PCR mixture by silicone oil. Reverse transcription followed by the first-round PCR was performed. Then, the second-round mixture was combined with first-round products by a centrifugation step followed by second round PCR during which fluorescence intensities were recorded and used for quantification. To establish the sensitivity of this novel assay a serial dilution of HCV reference standard was used. In plasma samples about 100 IU/ml HCV were consistently detected using the high pure viral RNA kit for nucleic acid purification. This detection limit was found to be about 20 fold increased compared with single-round RT-PCR and corresponded to 3.4 IU of HCV per capillary. Using a panel of HCV genotype standards the novel assay exhibited similar sensitivity for all HCV genotypes. The applicability for clinical routine testing was demonstrated by examining 156 clinical samples. Two-round RT-PCR with the LightCycler instrument using a single closed capillary throughout the procedure was found ideally suited for rapid (100 min), accurate and sensitive molecular diagnosis of active HCV infections. Since the capillaries remained closed during the procedure carry-over contamination was precluded.

Single-tube two-round polymerase chain reaction using the LightCycler instrument.

For many diagnostic applications, the specificity and sensitivity of polymerase chain reaction (PCR) is markedly enhanced by applying two rounds of PCR with nested or semi-nested pairs of primers. In two-round PCR protocols on the LightCycler instrument, amplification products must be collected from the capillaries by centrifugation, a procedure thought to be particularly prone to product carry-over. Development of a technique to perform two-round PCR with the LightCycler instrument in a single closed capillary. Silicone oil was used to separate the second-round primers from first-round PCR mixture during the first-round PCR. The feasibility of the principle was demonstrated using a semi-nested primer system for the PCR analysis of genomic DNA. The first-round PCR reaction mixture was loaded into the capillary and covered by oil. Then, the second-round PCR reaction mixture was layered on top of it. PCR was run in two rounds separated by a centrifugation step that combined the second-round PCR mixture with the first-round products. Amplified products were visualized by fluorescence melting curve analysis. When a dilution series of genomic DNA was used for the single-capillary two-round PCR, 0.1 ng of DNA could consistently be detected. This was a 10-fold increase of sensitivity in comparison with single-round PCR. With the new technique, the first-round reaction mixture was sufficiently separated from second-round primers by the oil layer. Two-round PCR on the LightCycler using a single closed capillary excluded the possibility of amplification product carry-over. This new technique can easily be adapted for numerous applications, and should show feasibility for many nested primer PCR applications currently in use to the clinical detection of virus-derived DNA.

Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects.

A great majority of patients seeking preimplantation genetic diagnosis (PGD) are women >35 years of age. In addition to being carriers for single gene defects, these women also have a higher risk of having children with Down's syndrome (trisomy 21). For these patients, it would be advantageous if a diagnostic test for trisomy 21 was developed, which could be used in conjunction with tests for single gene defects. Here, we assessed the feasibility of developing an accurate genetic test for diagnosing trisomy 21 and the mutation causing spinal muscular atrophy (SMA) in single cells using multiplex fluorescence polymerase chain reaction (PCR). Single- and two-round PCR were developed using a combination of primers for the survival motor neuron (SMN) gene exons 7 and 8 and two chromosome 21 short tandem repeats (STRs), D21S226 and D21S11. After only 36 cycles, 88 and 68% of normal single cells were screened for SMA mutations and trisomy 21 respectively. In multiplex PCR using only two primers (SMN exon 7 and D21S11) instead of four, the efficiency of SMA diagnosis was increased to 93%. In the same reactions, the D21S11 alleles were detected in 83% of the normal single cells. Clinical applications of this assay should enable detection of those embryos that have inherited three heterozygous alleles and, therefore, benefit many PGD patients who are at an increased risk of Down's syndrome.

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome.

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR-1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.

Genetics: Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and ΔF508 mutations*

W1282X (W) and delta F508 (delta) are the two most common mutations of the cystic fibrosis Israeli population. Patients who are homozygotes (WW and delta delta) as well as compound heterozygotes (W delta) present a severe phenotype of the disease. In the present study, we have developed a polymerase chain reaction (PCR)-based method for the detection of both mutations simultaneously in a single blastomere. Unfertilized human oocytes and single polyspermic blastomeres were subjected to a two-round PCR amplification: a first round of multiplex PCR followed by a second round of nested PCR, done separately at each locus. Clear signals at both loci were obtained in 51% (47/65) of oocytes and 69% (24/35) of blastomeres. The genotype of the single cell analysed was determined by endonuclease digestion of the W products and by heteroduplex formation of the delta F products. This diagnostic system will allow the identification of affected embryos (WW, delta delta, W delta) as well as phenotypically normal carriers (W+, +delta), and therefore may be used for cystic fibrosis preimplantation diagnosis in families who carry either or both mutations.