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Methods in Molecular Biology (Clifton,... 2015Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional...
Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.
Topics: DNA Primers; Real-Time Polymerase Chain Reaction; Software
PubMed: 25697660
DOI: 10.1007/978-1-4939-2365-6_13 -
Cold Spring Harbor Protocols Feb 2019The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates...
The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with DNA polymerase.
Topics: Base Composition; Buffers; DNA; DNA Primers; Nucleic Acid Conformation; Polymerase Chain Reaction; Taq Polymerase
PubMed: 30710022
DOI: 10.1101/pdb.prot095141 -
Methods in Molecular Biology (Clifton,... 2015Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the...
Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.
Topics: DNA Primers; Polymerase Chain Reaction
PubMed: 25697657
DOI: 10.1007/978-1-4939-2365-6_10 -
Nucleic Acids Research Jun 2017DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly...
DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
Topics: Animals; Bone and Bones; DNA; DNA Ligases; DNA Primers; DNA, Single-Stranded; Fossils; Gene Library; High-Throughput Nucleotide Sequencing; Horses; Humans; Liver; Nucleic Acid Hybridization; Oligonucleotides; Polymerase Chain Reaction; Sequence Analysis, DNA; Swine
PubMed: 28119419
DOI: 10.1093/nar/gkx033 -
Methods in Molecular Biology (Clifton,... 2023Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction...
Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction (PCR) with two competing allele-specific primers and a SNP specific common reverse primer. Sequence information flanking SNP of interest and fluorescent plate reader for end-point measurement or qPCR machine for real time measurement are required for the execution of the Amplifluor assay. In this chapter, the principle and working protocol of the Amplifluor assay based on end-point fluorescence detection of SNP allele is presented with an example.
Topics: Genotype; Polymorphism, Single Nucleotide; Polymerase Chain Reaction; DNA Primers; Alleles
PubMed: 36781643
DOI: 10.1007/978-1-0716-3024-2_13 -
Nature Communications Apr 2022One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of...
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
Topics: Algorithms; DNA Primers; Likelihood Functions; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35410464
DOI: 10.1038/s41467-022-29500-4 -
TSitologiia I Genetika 2017MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDRinsertionspecific flanking...
MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDRinsertionspecific flanking sequences (MuDRFs), it will be crucial for using Mu elementmediated mutants. The MuDRTAILPCR system was constructed and optimized using a combination of MuDRTIRnested specific primers and 12 arbitrary degenerate (AD) primers, modified reaction system and procedure and mutant DNA templates of 87 genotypes from M2 or M2:3 families created by crossing the W22::Mu line (active MuDR donor parent) from the UniformMu population with the Zong31 (Z31) line (recipient parent). Here 129 different MuDRFs were acquired by MuDRTAILPCR, accounting for 86.60 % of the total mutantspecific agarose gel bands. In addition, we confirmed the authenticity of the nonredundant flanking sequence amplifications. The amplified nonredundant flanking sequences accounted for 65.12 % of the total MuDRFs, and 88.00 % of the nonredundant MuDRFs were inserted inside the genes. These results show that the MuDRTAILPCR system that we developed can be used for specifically isolating MuDRFs.
Topics: Base Sequence; Crosses, Genetic; DNA Primers; DNA Transposable Elements; Gene Expression Regulation, Plant; Genes, Plant; Genotype; Mutagenesis, Insertional; Polymerase Chain Reaction; Zea mays
PubMed: 30484622
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2022Proximity ligation assay (PLA), also referred to as Duolink PLA technology, permits detection of protein-protein interactions in situ (<40 nm distance) at endogenous...
Proximity ligation assay (PLA), also referred to as Duolink PLA technology, permits detection of protein-protein interactions in situ (<40 nm distance) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and takes advantage of specific DNA primers covalently linked to the antibodies. A hybridization step followed by a PCR amplification with fluorescent probes then permits visualization of spots of proximity by fluorescence microscopy.
Topics: Antibodies; DNA Primers; Microscopy, Fluorescence; Protein Interaction Mapping; Proteins
PubMed: 34859407
DOI: 10.1007/978-1-0716-1948-3_13 -
BMC Ecology and Evolution May 2024Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for... (Comparative Study)
Comparative Study
Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.
Topics: Mollusca; Animals; DNA Barcoding, Taxonomic; DNA, Environmental; DNA Primers; Biodiversity
PubMed: 38822255
DOI: 10.1186/s12862-024-02265-8 -
PloS One 2023qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However...
AIMS
qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6.
METHODS
The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer.
RESULTS
Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition.
CONCLUSIONS
Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.
Topics: Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA; DNA Primers; Genomics
PubMed: 37083935
DOI: 10.1371/journal.pone.0284538