Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Primers”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed2006

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types.

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.

DNA Primers↗
PubMed1993

Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration.

The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.

Animals↗
PubMed1998

Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase.

The binding of bacteriophage T4 DNA polymerase (T4 pol) to primer-template DNA with 2-aminopurine (2AP) located at the primer terminus results in the formation of a hyperfluorescent 2AP state. Changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at the exonuclease and statically quenched polymerase sites. In the absence of Mg2+, a hydrophobic exonuclease site dominates over the polymerase site for possession of the primer terminus. The fractional concentration of primer termini in the exonuclease site was found to be 64 and 84% for correct (AP-T) and mismatched (AP-C) primer-templates, respectively. Exonuclease-deficient mutants, polymerase-switching mutants, and nucleoside triphosphates all shift this equilibrium toward the polymerase site. Synthesis of stereospecific hydrolysis resistant phosphorothioate 2AP-labeled DNA allowed Mg2+ ion binding titrations to be performed in the presence of bound DNA without the complication of the excision reaction. High- and low-affinity Mg2+ binding sites were observed in the presence of bound double-stranded (ds) DNA, with dissociation constants in the micromolar (WT Kd = 5.1 microM) and millimolar (WT Kd = 2.5 mM) concentration ranges. Mg2+ binding was found to be a key "conformational switch" for T4 pol. As the high-affinity Mg2+ binding sites are filled, the primer terminus migrates from the exonuclease site to a highly based stacked polymerase active site. Filling the low-affinity Mg2+ sites further shifts the primer terminus into the polymerase site. As the low-affinity Mg2+ sites are filled, T4 pol "loosens its grip" on the primer terminus, as shown by a large amplitude increase in the nanosecond rotational mobility of 2AP within the bound T4 complex. The hyperfluorescent exonuclease site is spatially localized to 2AP positioned on the primer end. The penultimate (n - 1) position, as well as n - 2 and n - 5 positions, reveals no detectable fluorescent enhancement upon binding. The observed position-dependent fluorescence data, when combined with time-resolved total-intensity and anisotropy data, suggest that the creation of the hyperfluorescent state is caused by phenylalanine 120 (F120) of T4 pol intercalating into 2AP primers much like that observed for phenylalanine 123 of RB69 DNA polymerase intercalating into deoxythymidine primers [Wang, J., et al. (1997) Cell 89, 1087-1099]. As Mg2+ binds in the exonuclease site of T4 pol, the primer terminus appears to be "pulled backward" into the active site, decreasing the concentration of F120-intercalated primer termini, and bringing the exonuclease active site residues closer to the primer terminus scissile phosphate bond.

2-Aminopurine↗
PubMed2004

A primer-dependent polymerase function of pseudomonas aeruginosa ATP-dependent DNA ligase (LigD).

Pseudomonas aeruginosa encodes two putative DNA ligases: a classical NAD(+)-dependent DNA ligase (LigA) plus an ATP-dependent DNA ligase (LigD). LigD exemplifies a family of bacterial proteins that consist of a ligase domain fused to flanking domains that resemble nucleases and/or polymerases. Here we purify LigD and show that it possesses an intrinsic polymerase function resident within an autonomous C-terminal polymerase domain, LigD-(533-840), that flanks an autonomous DNA ligase domain, LigD-(188-527). Native LigD and the polymerase domain are both monomeric proteins. The polymerase activity is manifest in three ways: (i) non-templated nucleotide addition to a blunt-ended duplex DNA primer; (ii) non-templated addition to a single-stranded DNA primer; and (iii) templated extension of a 5'-tailed duplex DNA primer-template. The divalent cation cofactor requirement for non-templated and templated polymerase activity is satisfied by manganese or cobalt. rNTPs are preferred over dNTPs as substrates for non-templated blunt-end addition, which typically entails the incorporation of only 1 or 2 nucleotides at the primer terminus. Templated dNMP addition to a 5'-tailed substrate is efficient with respect to dNTP utilization; the primer is elongated to the end of the template strand and is then further extended with a non-templated nucleotide. The polymerase activity is abolished by alanine substitution for two aspartates (Asp-669 and Asp-671) within the putative metal-binding site. We speculate that polymerase activity is relevant to LigD function in nonhomologous end-joining.

Alanine↗
PubMed1994

Evolutionary role of abortive transcript as a primer for DNA replication.

Abortive cycling features transcription initiation by RNA polymerase in both prokaryote and eukaryote. It is known that T7 RNA polymerase produces abortive transcripts up to eight ribonucleotides in length depending on the initial sequence of the DNA message. On the other hand, T7 RNA polymerase initiates DNA replication from the T7 primary origin by synthesizing primers. And the shortest primer from the phi l.lB promoter in the primary origin also seems to be eight ribonucleotides in length. Therefore, it is likely that the longest abortive transcript serves as the shortest primer for T7 DNA replication from the primary origin. Considering that promoters often exist in DNA replication origins for example, E. coli oriC and many eukaryotic origins, the early DNA replication system appears to have taken advantage of the abortive cycling of RNA-dependent RNA polymerase that already existed before the emergence of DNA world. The evolutionary primitive RNA polymerase could do both transcription and priming of DNA replication. Accordingly, abortive cycling would play an important role in evolution at the emergence of DNA world. The priming activity of the primitive RNA polymerase would be taken over by primase later, which seems to be a specialized RNA polymerase for abortive cycling.

Biological Evolution↗
PubMed1998

Development of long PCR techniques to analyze deletion mutations of the human hprt gene.

DNA primers and reaction conditions for long polymerase chain reaction amplification (PCR) of portions of the human hprt gene are presented. Use of these primers with DNA from previously defined hprt deletion mutants demonstrated production of the expected smaller size products as compared to DNA from wild type cells. These primers will be of value in the rapid analysis and subsequent sequencing of hprt deletion mutations in human cells.

Base Sequence↗
PubMed1993

Theileria parva: detection of genomic polymorphisms by polymerase chain reaction amplification of DNA using arbitrary primers.

Genomic DNA fingerprints were generated from Theileria parva piroplasm DNA by polymerase chain reaction amplification using arbitrary decamers as primers. Four of 12 primers tested generated amplified fragments which were polymorphic between different T. parva stocks or clones. Hybridization of radiolabeled total amplification products to Southern blots of T. parva piroplasm and uninfected bovine DNA confirmed that none of the amplified fragments were derived from any contaminating bovine DNA which may have been present in small quantities. Polymorphic DNA fragments were reproducibly amplified from independent DNA preparations made from cloned parasites. Individual amplified DNA fragments were gel purified and shown to hybridize to size-polymorphic EcoRI fragments in T. parva genomic DNA. Hybridization of individual amplified fragments to total fingerprints, generated using the same arbitrary primer, demonstrated that a single primer detected at least three independent genomic polymorphisms in T. parva DNA.

Animals↗
PubMed1980

A versatile primer for DNA sequencing in the M13mp2 cloning system.

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.

Base Sequence↗
PubMed1986

Mapping transcription start points on cloned genomic DNA with T4 DNA polymerase: a precise and convenient technique.

We have developed a precise and convenient mapping technique for determining transcription start points (tsp) on cloned genomic DNA using T4 DNA polymerase. This method uses single-stranded (ss) M13 DNA and therefore is, unlike S1 and Exo VII nuclease mapping methods, independent of the restriction endonuclease sites present in the insert. Essentially the protocol involves the following steps: hybridizing an mRNA to an ss M13 vector containing an antisense genomic DNA sequence spanning the presumptive tsp (cap site); annealing a DNA primer (M13 sequencing primer) to the M13 DNA at a site on this DNA upstream from the 5' end of the mRNA on the template DNA; extending the DNA primer with T4 DNA polymerase towards the 5' end of the mRNA. Since T4 DNA polymerase will not displace the mRNA: DNA hybrid, synthesis is blocked at the first nucleotide of the mRNA molecule. The length of the extended DNA products can then be determined with single nucleotide resolution on denaturing sequencing gels in parallel with a sequencing ladder. We have used this approach to map the tsp of the mouse skeletal alpha-actin gene. The sensitivity of the method allows precise mapping of transcripts present as 0.02-0.05% of the total RNA. This method is particularly valuable for mapping the tsp of genes which are known to contain a large intron between the first and second exons. It can also be applied to map the 5' border of any given exon of a gene in an M13 vector or in other vectors that give ss DNAs.

Actins↗
PubMed1996

Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.

Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.

Base Sequence↗
PubMed1999

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Autoanalysis↗
PubMed1995

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.

Animals↗
PubMed1993

Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation.

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosomes, maintaining telomere length and stabilizing chromosome ends. In vitro, telomerase from the ciliate Tetrahymena elongates single-stranded, guanosine-rich DNA primers by adding repeats of the Tetrahymena telomeric sequence, dT2G4. We have identified two activities of Tetrahymena telomerase in addition to the previously described processive elongation reaction: a 3'-5' nucleolytic cleavage of primer or product DNA and a nonprocessive mode of elongation. The nucleolytic cleavage activity removed residues not conforming to the telomeric repeat sequence from a primer 3' end, eliminating mismatch between DNA primer and RNA template sequences. Template-matched residues were also cleaved from primer or product DNA. Specific primer lengths, sequences, and concentrations stimulated cleavage and processive or nonprocessive elongation differentially. These newly identified activities suggest that telomerase may catalyze a range of telomere synthesis and repair functions and suggest mechanistic similarities between telomerase and RNA polymerase enzymes. On the basis of our results, we propose a model for telomerase primer binding, cleavage, and elongation.

Animals↗
PubMed1996

Substrate properties of fluorescent ribonucleotides in the terminal transferase-catalyzed labeling of DNA sequencing primers.

Terminal deoxynucleotidyltransferase (terminal transferase, E.C. 2.7.7.31) has been used to add a single fluorescent ribonucleotide to the 3' terminus of DNA sequencing primers, thereby creating primers suitable for automated DNA sequence analysis. The previously introduced procedure using fluorescein-UTP for the postsynthetic labeling of primers can, under appropriate reaction conditions, now be extended to commercially available fluorescein-ATP and fluorescein-CTP permitting greater flexibility in primer design. The products of these addition reactions have been shown to provide sequence data qualitatively and quantitatively identical to those obtained with conventional 5'-terminally labeled primers using cycle sequencing conditions in conjunction with an automated sequencer. Ribonucleotide derivatives of four other dyes (coumarin, tetramethylrhodamine, lissamine and Texas Red) were also examined for their potential in the terminal transferase-catalyzed reaction. Whereas coumarin-UTP was efficiently incorporated giving a monoaddition product, the conjugates of all other dyes with ATP, CTP and UTP were extremely poor substrates under all conditions tested.

Coumarins↗
PubMed2002

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene.

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.

Aged↗
PubMed1977

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers.

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription.

Avian Myeloblastosis Virus↗