Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta
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ABSTRACT:
SUBMITTER: Yumoto K
PROVIDER: S-EPMC10926516 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
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Project description:In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.
| S-EPMC5648668 | biostudies-literature
Project description:Cicadidae periostracum (CP), the medicinal name of cicada exuviae, is well-known insect-derived traditional medicine with various pharmacological effects, e.g., anticonvulsive, anti-inflammatory, antitussive, and anticancer effects; it is also beneficial for the treatment of Parkinson's disease. For appropriate CP application, accurate species identification is essential. The Korean pharmacopoeia and the pharmacopoeia of the People's Republic of China define Cryptotympana atrata as the only authentic source of CP. Species identification of commercially distributed CP based on morphological features, however, is difficult because of the combined packaging of many cicada exuviae in markets, damage during distribution, and processing into powder form. DNA-based molecular markers are an excellent alternative to morphological detection. In this study, the mitochondrial cytochrome c oxidase subunit I sequences of C. atrata, Meimuna opalifera, Platypleura kaempferi, and Hyalessa maculaticollis were analyzed. On the basis of sequence alignments, we developed sequence-characterized amplified-region (SCAR) markers for efficient species identification. These markers successfully discriminated C. atrata from the three other cicada species, and detected the adulteration of market CP samples. This SCAR assay is a rapid, simple, cheap, reliable, and reproducible method for species identification, regardless of sample form and status, and contributes to CP quality control.
| S-EPMC7143061 | biostudies-literature
Project description:BackgroundWe previously developed a simple method termed HpaII-McrBC PCR (HM-PCR) to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs) on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity.FindingsWe developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR) that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods.ConclusionsHM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.
| S-EPMC3127961 | biostudies-literature
Project description:Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3'-->5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10(7)-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
| S-EPMC2632903 | biostudies-literature
Project description:Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
| S-EPMC5593185 | biostudies-literature
Project description:DNA damage blocks DNA polymerase progression and increases miscoding. In this study, we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-Oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the mean modified efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.
| S-EPMC1945218 | biostudies-literature
Project description:BackgroundMultiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents.ResultsIn this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA.ConclusionA wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.
| S-EPMC2759926 | biostudies-literature
Project description:Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.
| S-EPMC4896431 | biostudies-other
Project description:PREMISE OF THE STUDY:Microsatellite markers were isolated in the rainforest tree Lophira alata (Ochnaceae), an important timber tree from Central Africa, and cross-amplified on its savannah counterpart, L. lanceolata. METHODS AND RESULTS:From a microsatellite-enriched library sequenced on a 454 GS FLX platform, 13 primer combinations were identified. Amplification was optimized in two multiplex reactions. The primers amplified di- and trinucelotide repeats, with two to seven alleles per locus. Eleven primers also amplified in L. lanceolata. CONCLUSIONS:Microsatellite primers developed for the genus Lophira displayed sufficient variation to investigate hybridization between congeneric species in the rainforest-savannah transition.
| S-EPMC4610311 | biostudies-literature
Project description:Coccidioides DNA was amplified from serum by a PCR using coccidioid-specific primers. A 239-bp product was visualized when 10 fg of exogenous coccidioidal DNA was subjected to amplification. This product was demonstrated in some human and mouse sera prior to the detection of coccidioidal antibodies.
| S-EPMC404645 | biostudies-literature