Thread-powered cell lysis and isotachophoresis: unlocking microbial DNA for diverse molecular applications.

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection.

Isopropanol-promoted DNA extraction by polydopamine functionalized magnetic particles based on metal coordination.

High-quality DNA is an important guarantee to start downstream experiments in many biological and medical research areas. Magnetic particle-based DNA extraction methods from blood mainly depend on electrostatic adsorption in a low-pH environment. However, the strong acidic environment can influence the DNA stability. Herein, a polydopamine-functionalized magnetic particle (PDA@Fe3O4)-based protocol was developed for DNA extraction from whole blood samples. In the protocol, Mg2+ and Ca2+ were utilized to bridge the adsorption of DNA by PDA@Fe3O4 via the metal-mediated coordination. Isopropanol was found to efficiently promote DNA adsorption by triggering the change of the conformation of DNA from B-form to more compact A-form. In 50 % isopropanol solution, the DNA adsorption efficiency was nearly 100 % in the presence of 0.5 mM Ca2+ or 1.5 mM Mg2+. The role of metal ions and isopropanol in DNA adsorption was explored. The protocol averts the strong acidic environment and PCR inhibitors, such as high concentrations of salt or polyethylene glycol. It demonstrates superiority in DNA yield (59.13 ± 3.63 ng µL-1) over the commercial kit (27.33 ± 4.98 ng µL-1) and phenol-chloroform methods (37.90 ± 0.47 ng µL-1). In addition, to simplify the operastion, an automated nucleic acid extraction device was designed and fabricated to extract whole genomic DNA from blood. The feasibility of the device was verified by extracting DNA from cattle and pig blood samples. The extracted DNA was successfully applied to discriminate the beef authenticity by a duplex PCR system. The results demonstrate that the DNA extraction protocol and the automated device have great potential in blood samples.

Treatment of long-term stored DNA--comparison between different methods to obtain high-quality material.

Long-term stored DNA can be sometimes the only source of genetic material of an organism that does not exist anymore, but a research interest still persists. However, there is a lack of information about useful methods to improve quality from such type of material. In this study, we compared four different protocols using DNA samples collected in 1998. Fresh DNA was also tested aiming to check the differences between these two material types. Sixteen samples of each DNA type treated with phenol-chloroform with PEG 5.0%, silica-gel membrane spin column, PEG 7.5%, and glass-fiber matrix spin column were submitted to spectrophotometer measurements, electrophoresis, PCR, and RFLP-PCR to assess the best method concerning yield, quality, and purity. Based on the results, purification with PEG 7.5% was considered the best method to treat aged DNA samples. In addition to the efficiency, this protocol has low cost. Analyzing the data, we also conclude that long-term stored DNA may be considered a reliable and potential resource for future molecular studies.

DNA Purification from Bloodstains and Buccal Cells/Saliva on FTA® Cards.

FTA® cards enable efficient, long-term storage of blood and buccal cells/saliva samples for future forensic DNA analysis; these are typically collected as known reference samples, as opposed to evidentiary, crime scene samples. Upon contact with the FTA® card, cells are lysed and the DNA is immobilized. Different FTA® cards are available and have been specially formulated based on sample type: bloodstains are added to the traditional FTA® Card, while colorless sources (e.g., buccal cells/saliva) are added to the FTA® Indicating Card. The main difference between these cards is the presence of a pink dye embedded in the indicating cards that becomes white when exposed to colorless fluids, like saliva; this aids in location confirmation of the stain for future sampling. Although DNA can be eluted/extracted from FTA® punches using various methods or, alternatively, direct STR amplification from unpurified punches can be performed, the protocol herein describes a simple purification method for bloodstained punches from FTA® Cards as well as buccal/saliva-stained punches from FTA® Indicating Cards. Following this purification, STR amplification can be performed via the "punch-in" method.

Low Volume STR Amplification Options: Coupling with Standard or Fast PCR, Traditional or Normalized DNA Extraction, and/or Traditional or Alternative Capillary Electrophoresis.

STR amplification leads directly to profile development, which is also impacted by DNA extraction and capillary electrophoresis detection. Amplification for forensic human identification purposes is inherently a costly process; reduced volume reactions have long been an effective cost-savings measure. Processing known, high-quality, single-source DNA samples (i.e., buccal samples) allows for the use of even lower reaction volumes. This chapter provides examples of 3 µL and 6 µL reactions for a variety of commercial amplification kits for use with buccal samples, including standard and fast PCR using KAPA2G™ Multiplex Mix. These reactions can be utilized with traditional DNA extracts or those obtained from a normalized extraction with the ChargeSwitch® Forensic DNA Purification Kit. They can be detected via traditional capillary electrophoresis using POP-4™ polymer and a 36 cm array, or an alternative method using POP-6™ polymer and a 22 cm array on the 3130 series Genetic Analyzer instruments. This chapter also includes protocols for the normalized extraction and alternative detection method.

Plasma micro-nanotextured polymeric micromixer for DNA purification with high efficiency and dynamic range.

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 108 cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis.

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Dye affinity cryogels for plasmid DNA purification.

The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9µmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA.