Extraction of Trace Amount of Severely Degraded DNA

2009 ◽  
Vol 64 (7-8) ◽  
pp. 581-589 ◽  
Author(s):  
Pin Lv ◽  
Xiangshan Zhou ◽  
Jinhua You ◽  
Bang-Ce Ye ◽  
Yuanxing Zhang
Keyword(s):  
Proteinase K ◽  
Degraded Dna ◽  
Dna Fragments ◽  
Dna Yield ◽  
Equus Asinus ◽  
Spin Column ◽  
Purification System ◽  
Dna Fragment ◽  
Yield And Purity ◽  
Colla Corii Asini

DNA extraction from food is always problematic especially from highly processed samples which contain only trace amounts of severely degraded DNA fragments. In this work, to extract trace amounts of small DNA fragments of the traditional Chinese medicine (TCM) colla corii asini derived from highly processed Equus asinus skin, three strategies were compared for its authentication. With some optimizations, the modified QIAquick spin column method achieved higher DNA yield and purity in comparison with the “SDS/proteinase K” method and the “Wizard magnetic DNA purification system for food” method. Further studies showed that at least 0.4 g colla corii asini was needed to obtain enough DNA extracts for PCR-based detection by the method and only amplicons of less than 100 bp could be generated from the DNA extracts which confirmed the efficiency of the method in small DNA fragment extraction. The DNA obtained by this method was suitable to be used in PCR-based authentications.

scholarly journals Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 146
Author(s):  
Catarina Xavier ◽  
Mayra Eduardoff ◽  
Barbara Bertoglio ◽  
Christina Amory ◽  
Cordula Berger ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Extraction Method ◽  
Fragment Size ◽  
Molecular Genetic ◽  
Extraction Methods ◽  
Degraded Dna ◽  
Size Analysis ◽  
Dna Fragments ◽  
Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

scholarly journals An efficient DNA extraction method from bone and tooth samples by complete demineralization followed by the use of silica-based columns

2014 ◽  
Vol 23 (2) ◽  
pp. 101-107 ◽  
Author(s):  
Md Mahamud Hasan ◽  
Tania Hossain ◽  
Ashish Kumar Majumder ◽  
Pilu Momtaz ◽  
Tarana Sharmin ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Extraction Method ◽  
Mechanical Grinding ◽  
High Concentration ◽  
Building Collapse ◽  
Dna Yield ◽  
Dna Extraction Method ◽  
Spin Column ◽  
Cryogenic Method ◽  
Dna Profiles

A highly efficient strategy for recovery of genomic DNA from bone and tooth samples is presented by complete demineralization of the bone pieces or intact tooth with high concentration of EDTA followed by spin column treatment without the need of mechanical grinding or cryogenic method for pulverizing the samples. The DNA yield was between 8 and 12 ng/?l from approximately 1 ?2 g of the starting material. Completed DNA profiles were obtained from of all the bones (52) and tooth (270) samples received from the unidentified victims from a recent building collapse, the Rana Plaza disaster in Dhaka, Bangladesh. DOI: http://dx.doi.org/10.3329/dujbs.v23i2.20089 Dhaka Univ. J. Biol. Sci. 23(2): 101-107, 2014

scholarly journals Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

2019 ◽  
Vol 8 (11) ◽  
pp. 1995 ◽  
Author(s):  
Moon ◽  
Shin ◽  
Kim ◽  
Lee ◽  
Mankhong ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Proteinase K ◽  
High Yield ◽  
Clinical Samples ◽  
Diagnostic Biomarkers ◽  
Micro Rnas ◽  
Biomarker Research ◽  
Protein Rich ◽  
Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
Author(s):  
Jee-Soo Lee ◽  
Miyoung Kim ◽  
Moon-Woo Seong ◽  
Han-Sung Kim ◽  
Young Kyung Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Analytical Sensitivity ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specimen Type ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Ctdna Analysis ◽  
Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

scholarly journals MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

2020 ◽  
Vol 36 (11) ◽  
pp. 3322-3326
Author(s):  
Michael Schwarz ◽  
Marius Welzel ◽  
Tolganay Kabdullayeva ◽  
Anke Becker ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Web Application ◽  
De Novo ◽  
Supplementary Information ◽  
Dna Fragments ◽  
Synthetic Dna ◽  
Custom Made ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Simple Silica Column–Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues

Cartilage ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 417-427 ◽  
Author(s):  
Whitaik David Lee ◽  
Rahul Gawri ◽  
Toshikazu Shiba ◽  
Ae-Ri Ji ◽  
William L. Stanford ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Proteinase K ◽  
Inorganic Polyphosphates ◽  
Column Method ◽  
Spin Column ◽  
Mammalian Tissues ◽  
Biological Sources ◽  
Low Levels

Objective. Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. Design. Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4′,6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. Results. Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10−10 mol. Conclusions. This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.

Assessment of DNA Yield and Purity: an Overlooked Detail of PCR Troubleshooting

2012 ◽  
Vol 34 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Kelly A. Boesenberg-Smith ◽  
Mohammad M. Pessarakli ◽  
Donna M. Wolk
Keyword(s):  
Dna Yield ◽  
Yield And Purity

scholarly journals Identifikasi dan isolasi promoter gen pembungaan kakao TcLFY Identification and isolation for promoter of TcLFY cacao flowering gene

2016 ◽  
Vol 75 (1) ◽  
Author(s):  
Djoko SANTOS ◽  
Agustina A. HANDAYAN ◽  
Sukarti MOELJOPAWIRO
Keyword(s):  
Input Data ◽  
Open Reading Frame ◽  
Genome Walking ◽  
Dna Fragments ◽  
Reading Frame ◽  
Dna Fragment

SummaryPromoter is a regulator of geneexpression for a phenotype or trait carried bythe gene. In the structure, a promoter locatedbeyond the 5’ end of the open reading frame ofthe gene on which its expression is regulated.This research aimed to isolate the DNAfragment flanking TcLFY at the 5’ end and toanalyze whether the fragment has charac-teristics of the promoter, primarily the coremotifs of promoter. Using Genome Walkingtechnique, DNA fragments flanking the TcLFYgene at its 5’ end was isolated. Analysis of theDNA sequence was done using onlinecomputer software accessible through web sitewww.softberry.com and an entry sequence ofthe flanking DNA fragment along with the 2.5kb TcLFY sequence. The result indicated thatthe flanking fragment has core motifs for apromoter at proper positions, which are TATAbox at position –80, CAT boxes (CCAAT) at -387 and –626, and GC boxes that are known asUAS were found at the -323 and –537positions. To obtain a conclusive result, thispromoter sequence needs to be furtherexamined to confirm its function.RingkasanPromoter merupakan pengendali ekspresigen untuk memunculkan fenotipe atau karakteryang dibawa oleh gen tersebut. Di dalamstrukturnya, promoter umumnya terletak didaerah ujung 5’ gen yang dikendalikanekspresinya. Tujuan penelitian ini adalahmendapatkan fragmen DNA yang mengapitgen pengendali pembungaan kakao (TcLFY)dan menganalisisnya apakah memilikikarakteristik promoter, yaitu mengandungmotif-motif inti (core motifs) dari promoter.Dengan teknik Genome Walking, fragmenDNA pengapit gen TcLFY di ujung 5’ dapatdiisolasi. Analisis sekuen menggunakanperangkat lunak komputer online (www.softberry.com) dengan input data fragmentersebut ditambah gen TcLFY 2,5 kb dibawahnya, mengindikasikan adanya beberapamotif inti promoter pada posisi yang sesuai,yaitu kotak TATA pada lokasi –80, kotak CAT(CCAAT) di posisi -387 dan –626, dan kotakGC yang merupakan UAS dijumpai padalokasi -323 dan –537. Untuk memperoleh hasilyang bersifat konklusif, sekuen promoter inimasih perlu diuji fungsinya.

scholarly journals Polymerase Chain Reaction Method to Detect Canis Materials by Amplification of Species-Specific DNA Fragment

2004 ◽  
Vol 87 (5) ◽  
pp. 1195-1199 ◽  
Author(s):  
Hong-Wei Gao ◽  
Bao-Liang Xu ◽  
Cheng-Zhu Liang ◽  
Yi-Bing Zhang ◽  
Lai-Hua Zhu
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Method ◽  
Degraded Dna ◽  
Effective Control ◽  
Heat Denaturation ◽  
Spongiform Encephalopathy ◽  
Chain Reaction ◽  
Animal Blood ◽  
Dna Fragment ◽  
Polymerase Chain

Abstract Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

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