Short Target Sequences Research Articles

InflammamiRs in focus: Delivery strategies and therapeutic approaches.

microRNAs (miRNAs) are small non-protein-coding RNAs which are essential regulators of host genome expression at the post-transcriptional level. There is evidence of dysregulated miRNA expression patterns in a wide variety of diseases, such as autoimmune and inflammatory conditions. These miRNAs have been termed "inflammamiRs." When working with miRNAs, the method followed, the approach to treat or diagnosis, and the selected biological material are very crucial. Demonstration of the role of miRNAs in particular disease phenotypes facilitates their evaluation as potential and effective therapeutic tools. A growing number of reports suggest the significant utility of miRNAs and other small RNA drugs in clinical medicine. Most miRNAs seem promising therapeutic options, but some features associated with miRNA therapy like off-target effect, effective dosage, or differential delivery methods, mainly caused by the short target's sequence, make miRNA therapies challenging. In this review, we aim to discuss some of the inflammamiRs in diseases associated with inflammatory pathways and the challenge of identifying the most potent therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics. We also discuss the status of inflammamiRs in clinical trials.

Short Activators and Repressors of RNA Toehold Switches.

RNA toehold switches are a widely used class of molecule to detect specific RNA "trigger" sequences, but their design, intended function, and characterization to date leave it unclear whether they can function properly with triggers shorter than 36 nucleotides. Here, we explore the feasibility of using standard toehold switches with 23-nucleotide truncated triggers. We assess the crosstalk of different triggers with significant homology and identify a highly sensitive trigger region where just one mutation from the consensus trigger sequence can reduce switch activation by 98.6%. However, we also find that triggers with as many as seven mutations outside of this region can still lead to 5-fold induction of the switch. We also present a new approach using 18- to 22-nucleotide triggers as translational repressors for toehold switches and assess the off-target regulation for this strategy as well. The development and characterization of these strategies could help enable applications like microRNA sensors, where well-characterized crosstalk between sensors and detection of short target sequences are critical.

Asymmetric stem-loop-mediated isothermal amplification of nucleic acids for DNA diagnostic assays by simple modification of canonical PCR primers.

Nucleic acid–based assays have been adopted as mainstream tools for clinical diagnostics, food safety, and environment monitoring with the merits of accuracy, rapidity, and sensitivity. Loop-mediated isothermal amplification (LAMP) is a well-established method to rapidly identify nucleic acids and has gained recognition and been developed for clinical applications in resource-limited areas. However, the needs for specifically designed primer sets and non-specific amplification hinder the development of LAMP-based nucleic acid tests. Here, a promoted method, termed asymmetric stem-loop–mediated isothermal amplification (ASLAMP) by simple modification of canonical PCR primers, was developed to attempt to overcome those drawbacks. The two primers in the ASLAMP reaction can be easily obtained by adding a stem-loop sequence part to one PCR primer at 5′-ends to get the folding primer (FP), then adding the same primer to the counter canonical PCR primer at 5′-ends to get the turn-back primer (TP). The ASLAMP method was demonstrated in detecting the H1N1 gene fragment with merits of simple primer design, short target sequence, and high amplification efficiency. In addition, the ASLAMP method showed similar efficacy compared with LAMP targeting at the same H1N1 gene sequence. Furthermore, Shigella detection monitored by real-time fluorescence and endpoint colorimetric approaches were taken as examples for evaluation of the practical application of the ASLAMP method, both offered 100% sensitivity and specificity. In conclusion, the novel ASLAMP method with simplicity of primer design, low requirement of equipment, efficiency, and rapidity has exhibited its great prospect for establishment of DNA isothermal amplification in point of care application.

Detection of chicken adulteration in beef via ladder-shape melting temperature isothermal amplification (LMTIA) assay

The ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technique with advantages of simple mechanism, easy primer design, quick turn-around time, high specificity, high sensitivity and short target sequence compared to the loop-mediated isothermal amplification (LAMP) technique. The objective of this research was to establish the LMTIA assay for detection of chicken in beef. The LMTIA primers targeting the prolactin receptor gene of Gallus gallus were designed, the LMTIA reaction system was optimized, the specificity and the sensitivity of the LMTIA assay were determined. Our results showed that the LMTIA assay was able to specifically detect 100 ng genomic DNA of Gallus gallus, without detecting the 100 ng genomic DNA of Anas platyrhynchos, Felis catus, Sus scrofa, Canis lupus familiaris, Bos taurus or Capra hircus. The sensitivity of the LMTIA assay was 100 pg genomic DNA of G. gallus. Furthermore, the LMTIA assay was able to detect the chicken adulterated in beef with ≥0.1% (w/w) detection limit (LOD). This study holds a promise for facilitation of the surveillance of the commercial adulteration of chicken in beef.

Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing.

It is important to control CRISPR/Cas9 when sufficient editing is obtained. In the current study, rational engineering of guide RNAs (gRNAs) is performed to develop small-molecule-responsive CRISPR/Cas9. For our purpose, the sequence of gRNAs are modified to introduce ligand binding sites based on the rational design of ligand–RNA pairs. Using short target sequences, we demonstrate that the engineered RNA provides an excellent scaffold for binding small molecule ligands. Although the ‘stem–loop 1’ variants of gRNA induced variable cleavage activity for different target sequences, all ‘stem–loop 3’ variants are well tolerated for CRISPR/Cas9. We further demonstrate that this specific ligand–RNA interaction can be utilized for functional control of CRISPR/Cas9 in vitro and in human cells. Moreover, chemogenetic control of gene editing in human cells transfected with all-in-one plasmids encoding Cas9 and designer gRNAs is demonstrated. The strategy may become a general approach for generating switchable RNA or DNA for controlling other biological processes.

A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.

Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5′-CGC GGC GCT ACA TAT TAG AC-3′, Reverse: 5′-GCA CTT AAC CGA CCG TCG AC-3′). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44–66% of cephalopod species, corresponding to about 310–460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268–271; Peters et al. 2015 Mar. Ecol. 36, 1428–1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.

A189 NON-ALCOHOLIC FATTY LIVER DISEASE AND SEN-V INFECTION: AN INTERIM ANALYSIS

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is a common liver disease in which 10–20% of patients are at risk of progressing to cirrhosis. Several risk factors and genetic variants have been proposed to explain such progression but these fail to explain all cases. SEN-V is a hepatotropic ssDNA virus transmitted by both parenteral and non-parenteral routes. In a previous study we documented serologic evidence of SEN-V infection in 21% of 108 patients with chronic Hepatitis B (HBV), C (HCV) or PBC disease. In a recent Japanese study, HCV patients who were SEN-V positive had higher rates of disease progression than those who were SEN-V negative. Conversely, in an Iranian study, liver enzyme levels were lower in HBV patients co-infected with SEN-V. No study has yet documented the prevalence or impact of SEN-V infection in NAFLD. Aims To determine the prevalence and impact of SEN-V infection in NAFLD. Methods Thirty-three consecutive patients with NAFLD confirmed on liver biopsy had serologic testing for SEN-V DNA using short and long primer target sequences; sequencing was confirmed by BLAST and phylogenetic analysis. Patients with other causes of chronic liver disease were excluded. ALT levels, NAFLD activity scores (NAS), and fibrosis scores on biopsy were compared between the SEN-V positive and negative groups using Mann-Whitney, t-test, and chi squared as appropriate. Results SEN-V serologic testing was positive in 15/33 (45%) NAFLD patients, significantly higher than the 24/108 (21%) reported in non-NAFLD historical controls (p < 0.01). Mean serum ALT levels were similar in the two cohorts (SEN-V positive: 109 +/- 163 versus SEN-V negative: 62 +/- 54 IU/L, p= 0.27). The median NAS scores were identical in both groups (5/8) 95% CI [3,6]. Although the median fibrosis stage in SEN-V positive patients was lower (1.0 95% CI [0,3] vs. 2.5 95% CI [1,4] respectively), the difference did not reach statistical significance (p= 0.27). Conclusions Within the limits of this small interim analysis, serologic evidence of SEN-V infection was more common amongst NAFLD patients than historical controls with non-NAFLD chronic liver diseases. However, there were no significant differences in ALT levels, NAS scores or fibrosis stages between SEN-V positive versus negative NAFLD patients. These results suggest that if SEN-V infection has any impact on NAFLD, it may contribute to the development but not severity of the disease. Funding Agencies None

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets.

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultrashort DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNVs) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. After sequencing on the Oxford Nanopore Technologies MinION platform, the resulting repeat sequences can be aligned to produce a highly accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences <100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of <10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA &lt;em&gt;In Situ&lt;/em&gt; Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

To Cleave or not to Cleave: Predicting the Target Specificity of CRISPR-Cas Systems through Theoretical Modeling

Many prokaryotes employ the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat – CRISPR associated) system to fend off attacks by hostile genetic elements. This adaptive immune system recognizes invaders based on their level of sequence complementarity with RNA transcribed from a library of past invasions stored at the CRISPR locus. To avoid infection, the CRISPR interference complex must be able to single out a short target sequence (20 – 40 nt) among a total of 105-106 nt in the cell, while at the same time recognizing targets that have evolved away from the record stored at the CRISPR locus. Despite the tremendous interest the CRISPR-Cas9 system has gained as a novel genome editing tool, the sequence preference of the interference complex remains poorly understood. Experiments have shown that it is not just the amount of mutations, but also their placement along the guide/target, that determine the level of interference.Through kinetic modeling we provide simple rules to assess sequence specificity based on mismatch patterns, and quantitatively explain the experimentally observed seed region over which no mismatches are permitted. Moving beyond sequence complementarity, we also quantify how changes in the conformation of the interference complex can serve to improve specificity during target recognition. Finally, by fitting our model to published experimental data, we give estimates for the base-pairing energies within the interference complex for various CRISPR systems.

Beyond Diffusion-Controlled Kinetics: How a Protein Implements a Fast Search on DNA to Get to its Specific Binding Site

There are a wide number of proteins that initiate and regulate the transcription of genes. All have DNA-binding domains that give them the ability to bind specific sequences. Classical binding reactions are controlled by diffusion limit. In those cases, association rate for binding doesn’t exceed 108 M−1S−1 and then, for high affinity binding (nM), dissociation rate would expands dozens of seconds. Find a short target sequence within the enormous pool provided by genomic DNA requires protein moving fast towards the specific binding site (fast association and dissociation while searching). Before reaching their targets, DNA-binding proteins encounter nonspecific DNA first and bind to it, although with weaker affinity than DNA specific sequence. As observed first in 1970, DNA binding proteins binds its target ∼100 times faster than allowed by the 3D diffusion limit. This faster-than-diffusion binding was explained by the facilitated-diffusion model, in which a DNA-binding protein interacts with nonspecific DNA before reaching its target by three main mechanism: sliding, where protein slides in 1D along nonspecific DNA; hopping, where the protein dissociates from DNA briefly, performing free 3D diffusion, and lands back on DNA at other close location; and jumping, where the protein's DNA landing location is not correlated to the dissociation site. Facilitate diffusion has been studied theoretically, computationally and experimentally. However, very little is known from the protein viewpoint, and how protein and DNA dialogue in an efficient manner. We are using the DNA-binding engrailed homeodomain module that binds with high affinity to DNA sequence TAATTA/G. In the present work we will analyze how DNA sequence seems to have evolved towards an organization that ensures biologically precise gene expression. Combination of specific sequence binding, degenerated sequence binding and nonspecific binding could promote an antenna effect for guiding the protein to the specific site. Mechanical statistical models help us to confirm the experimental data and shed light on this fundamental and multifactorial biological problem.

MiRNA-based therapies: strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents.

The discovery of miRNAs as important regulatory agents for gene expression has expanded the therapeutic opportunities for oligonucleotides. In contrast to siRNA, miRNA-targeted therapy is able to influence not only a single gene, but entire cellular pathways or processes. It is possible to supplement downregulated or non-functional miRNAs by synthetic oligonucleotides, as well as alleviating effects caused by overexpression of malignant miRNAs through artificial antagonists, either oligonucleotides or small molecules. Chemical oligonucleotide modifications together with an efficient delivery system seem to be mandatory for successful therapeutic application. While miRNA-based therapy benefits from the decades of research spent on other therapeutic oligonucleotides, there are some specific challenges associated with miRNA therapy, mainly caused by the short target sequence. The current status and recent progress of miRNA-targeted therapeutics is described and future challenges and potential applications in treatment of cancer and viral infections are discussed.

Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding.

Formalin fixation and paraffin embedding (FFPE) is a standard method for tissue sample storage and preservation in pathology archives. The Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a useful method for gene expression analysis, but its sensitivity is significantly decreased in FFPE tissue due to the fixation process. This process results in chemical modifications of RNA, cross-links proteins to RNA, and degrades RNA in these archived samples, hindering the reverse transcription step of the conventional RT-pPCR method and preventing generation of a cDNA that is long enough for the subsequent quantitative PCR step. In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides. Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3months to 4years, and was found to be comparable to results obtained with frozen liver tissues. Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue.

Manipulation of T cell function and conditional gene targeting in T cells.

Cre-Lox recombination is known as a site-specific recombinase technology, and is widely used to carry out modification at specific sites in the DNA of cells. The system consists of an enzyme, Cre recombinase, that recombines a pair of short target sequences, the lox sites. The Cre-Lox system can be used to activate or repress a gene depending on the placement of the lox sites. Placing the Cre recombinase under the control of a cell-specific promoter allows expression only in specific cells or cellular subsets, thus providing a powerful tool for analysis of gene function at specific developmental or physiological niches. Nowadays almost every aspect of T cell biology can be approached by a specific Cre model. This powerful tool allows scientists to overcome the limitations of gene-deficient animals and target a gene of interest specifically in T cell or T cell subsets by appropriate placement of the lox sites. Here we describe the main Cre lines that enable gene targeting in T helper cells or CD4 T cell subsets, and the most common methods of assessing the recombination efficiency.

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

Single-Molecule Observation of Viral DNA Targeting by CRISPR/Cas Immune Systems

Bacteria and archaea maintain a history of viral infections by integrating small fragments of foreign DNA into specialized genomic loci called clustered regularly interspaced short palindromic repeats (CRISPRs). An adaptive immune response is triggered during subsequent infections by CRISPR-derived RNAs (crRNAs), which function together with CRISPR-associated (Cas) proteins to identify and destroy complementary viral DNA sequences. DNA targeting by crRNA/Cas ribonucleoprotein surveillance complexes proceeds via RNA-DNA base-pairing interactions and requires melting of the double-stranded substrate, as well as protein-mediated recognition of a proximal DNA sequence motif to discriminate self from non-self. How these complexes find short target sequences within the larger context of genomic DNA is unknown, and prior studies have been limited by their use of oligonucleotide substrates and dependence on bulk electrophoretic mobility shift assays that obscure all dynamics of the search process. To gain deeper insights into this critical step of CRISPR/Cas based immunity, we are using fluorescence microscopy to visualize single crRNA/Cas complexes on nanofabricated curtains of viral DNA in real-time. These experiments enable direct observation of the kinetics and position-dependence of DNA binding, and have revealed the mechanism by which phylogenetically distinct surveillance complexes use sequence information in the crRNA to locate complementary DNA targets. Ongoing work is aimed at understanding how this recognition event promotes destruction of viral DNA by dedicated Cas nucleases.

CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI

Cytosine methylation promotes deamination. In eukaryotes, CpG methylation is thought to account for CpG underrepresentation. Whether scarcity of CpGs in prokaryotic genomes is diagnostic for methylation is not clear. Here, we report that Mycoplasms tend to be CpG depleted and to harbor a family of constitutively expressed or phase variable CpG-specific DNA methyltransferases. The very CpG poor Mycoplasma penetrans and its constitutively active CpG-specific methyltransferase M.MpeI were chosen for further characterization. Genome-wide sequencing of bisulfite-converted DNA indicated that M.MpeI methylated CpG target sites both in vivo and in vitro in a locus-nonselective manner. A crystal structure of M.MpeI with DNA at 2.15-Å resolution showed that the substrate base was flipped and that its place in the DNA stack was taken by a glutamine residue. A phenylalanine residue was intercalated into the "weak" CpG step of the nonsubstrate strand, indicating mechanistic similarities in the recognition of the short CpG target sequence by prokaryotic and eukaryotic DNA methyltransferases.

Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer

In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51 min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A = 2, AC = 1, B = 4, C = 3, D = 3, AE = 2, F = 1, BF = 2, G = 4) were amplified equally well with a similar threshold cycle (C t) number (22.9 ± 1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues.