Localized transfection on arrays of magnetic beads coated with PCR products

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.     

Localized transfection with magnetic beads coated with PCR products and other nucleic acids

The bead transfection method involves binding nucleic acids onto 3-microm-diameter paramagnetic beads, treating the beads with transfection reagent, and using them as scaffolds to direct transfection to individual cells or regions in a population. Typically, PCR products are used because they can be conveniently generated using biotinylated primers and can introduce site-directed mutations, without the need for cloning or plasmid purification. However, the method can be adapted to transfect plasmid DNA or RNA. The magnetic properties of the beads allows magnets to direct the loci of transfection in cell culture; magnetic arrays are built in cell culture chambers to allow multiple parallel transfections on the same microscope coverslip. The PCR reaction and transfection can be carried out in 1 d, and transfection results can be viewed in 24-48 h.     

Rapid preparation of single stranded DNA from PCR products by streptavidin induced electrophoretic mobility shift.

Streptavidin induced electrophoretic mobility shift was used to prepare single stranded (ss) DNA amplified with the polymerase chain reaction in the presence of a biotinylated and a non-biotinylated primer. A variety of denaturing conditions, including incubation at 95 degrees C in 50% formamide can be used without disrupting the streptavidin-biotinylated-ssDNA complex. Following electrophoresis, pure non-biotinylated DNA can be efficiently recovered from 7 M urea gels because it is well separated from the severely retarded streptavidin-biotinylated-ssDNA complex. Quantitative complexing of biotinylated ssDNA can occur at a streptavidin to DNA molar ratio of 1 or more.     

Presence of single-stranded DNA in PCR products of slow electrophoretic mobility.

PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.     

Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.     

Isolation of single-stranded DNA from loop-mediated isothermal amplification products.

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.     

Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

DNA helicases displace streptavidin from biotin-labeled oligonucleotides

Helicases are enzymes that use energy derived from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. The helicases used in this study, gp41 and Dda, are from the bacteriophage T4, an excellent system for studying enzymes that process DNA. gp41 is the replicative helicase and has been shown to form a hexamer in the presence of ATP. In this study, protein cross-linking was performed in the presence of either linear or circular single-stranded (ss) DNA substrates to determine the topology of gp41 binding to ssDNA. Results indicate that the hexamer binds ssDNA by encircling it, in a manner similar to that of other hexameric helicases. A new assay was developed for studying enzymatic activity of gp41 and Dda on single-stranded DNA. The rate of dissociation of streptavidin from various biotinylated oligonucleotides was determined in the presence of helicase by an electrophoretic mobility shift assay. gp41 and Dda were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP-dependent reaction. Helicase-catalyzed dissociation of streptavidin from the 3'-end of a biotin-labeled 62-mer oligonucleotide occurred with a first-order rate of 0.17 min-1, which is over 500-fold faster than the spontaneous dissociation rate of biotin from streptavidin. Dda activity leads to even faster displacement of streptavidin from the 3' end of the 62-mer, with a first-order rate of 7.9 s-1. This is more than a million-fold greater than the spontaneous dissociation rate. There was no enhancement of streptavidin dissociation from the 5'-biotin-labeled oligonucleotide by either helicase. The fact that each helicase was capable of dislodging streptavidin from the 3'-biotin label suggests that these enzymes are capable of imparting a force on a molecule blocking their path. The difference in displacement between the 5' and 3' ends of the oligonucleotide is also consistent with the possibility of a 5'-to-3' directional bias in translocation on ssDNA for each helicase.     

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.     

Negatively supercoiled DNA from plants infected with a single-stranded DNA virus

A method for isolating covalently closed circular double-stranded DNA from plants infected with the geminivirus, tomato golden mosaic virus, is described. Ethidium bromide titration showed this DNA to be negatively supercoiled with a superhelical density of -0.062. The presence of S1 nuclease-sensitive secondary structure in the supercoiled DNA was demonstrated by its conversion to the open circular and linear DNA forms on treatment with this enzyme.     

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental ¹³C/¹⁵N/²H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC and ¹³C₉/¹⁵N₂/²H_{(1′,2″,3′,4′,5′,5″)}-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective ²H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1′–H2′ and C2′–H2′ resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

磁珠法快速提取基因组DNA的实验研究

针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。     

Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips

A method for fluorescently labeled single-stranded DNA (ssDNA) production during single-stage polymerase chain reaction (PCR) for subsequent hybridization on a biochip was described. This approach, whose efficiency was confirmed in the case of DARC gene, is considered as an alternative to two-stage nested PCR, consisting of two separate reactions: symmetric and asymmetric. Implementation of PCR in a single stage was achieved due to the use of a truncated excess primer in the second stage that does not anneal on the matrix during the cycles of symmetric stage of PCR and that enters the reaction after decrease of the annealing temperature in asymmetric stage. As a result, high efficiency of genotyping by means of hybridization on biochips is maintained. The suggested approach will allow us to reduce the time, working hours, and risk of contamination when researching biochips.