Single-Tube DNA Purification and Cloning Using Ultrafiltration Devices
Total Page：16
File Type：pdf, Size：1020Kb
Scientific & Technical Report PN 33205 Single-tube DNA Purification and Cloning Using Ultrafiltration Devices Authors: Fang Qi1, Kevin Seeley2, and Timothy W. Short1 1 Biology Department, Queens College and the Graduate School of the City University of New York, 65-30 Kissena Boulevard, New York, NY 11367-1597 2 Pall Life Sciences, Scientific and Laboratory Services, 600 South Wagner Road, Ann Arbor, MI 48103-9019 Abstract The resulting ligation-ready DNA fragments were Traditional methods for subcloning DNA fragments ligated to digested vector directly within the or shuttling sequences between vectors involve ultrafiltration device, and then transformed into the ligation of restriction digest fragments from competent cells. Using Pall Life Sciences Nanosep chromosomes, plasmids, cosmids, or PCR products devices, we were able to achieve significant savings into specific sites on a selected cloning vector. In in time, labor, and starting materials, while the order to desalt, remove primers or adapters, or resulting yield of transformed recombinants concentrate DNA fragments, researchers generally roughly equaled that of the traditional techniques. use gel electrophoresis and precipitation steps that are labor-intensive and time-consuming. In Introduction an effort to develop a more streamlined process, Standard molecular cloning techniques require we compared traditional methods of subcloning enzymatic modification and purification steps to PCR-generated fragments against a rapid procedure prepare DNA sequences for ligation and using Nanosep® centrifugal ultrafiltration devices. transformation into bacterial cells. In the past, After optimizing the parameters for effective target procedural steps have included either serial or DNA purification, we synthesized PCR products simultaneous double restriction digests followed with terminal adapter sequences containing by gel electrophoresis to purify away adapter restriction sites. These adapters were digested, sequences. If not removed, the remaining adapter generating the appropriate cohesive ends for ligation. sequences compete for ligation sites and The resulting DNA products were concentrated significantly increase the number of spurious and desalted while simultaneously allowing the recombinants and reduce recombination primers and adapter fragments to pass into the efficiency. Because of the multiple purification discarded filtrate during a five-minute centrifugation steps with low recovery yields obtained with step (Figure 1). standard techniques, these methods require large amounts of starting DNA to guarantee sufficient Figure 1 vector and insert material to complete subsequent Directional Subcloning of PCR Products ligation and transformation steps (1). Synthesize PCR with Primers Containing Restriction Digest Sites Standard subcloning protocols incorporate a plasmid-based positive selection such as ampicillin resistance, allowing researchers to identify which Site A Site B colonies are transformed with vector sequences. Vector components such as the LacZ gene Cut and Remove Adapter A encoding the b-galactosidase enzyme can be A used to determine which antibiotic-resistant Cut and Remove Adapter B transformants are recombinant by blue/white A B screening in the presence of the X-gal substrate. Ligate and Transform Prepared Insert If an insert interrupts the polylinker in the LacZ, gene expression is disrupted and the ampicillin- resistant transformant will appear white. Typically, the first step in the transfer of a DNA sequence for A pool of PCR products was divided into two identical cloning involves digestion of the DNA by restriction enzymes aliquots. One fraction was precipitated with 1/10 volume 3 M to produce terminal sequence overhangs that facilitate ligation sodium acetate and 2 volumes ethanol, chilled at -20 °C for into a similarly prepared vector. Following each digestion step, 2 hr, centrifuged at high speed (12,000 x g) for 30 min, rinsed any compatible adapter sequences need to be removed so they with 70% ethanol, and air dried prior to resuspension in 40 µL will not compete with the desired insert during subsequent sterile distilled H2O (sdH2O). The second fraction was diluted ligation. The most frequently used method for this purification to 500 µL and centrifuged at 5,000 x g for 15 min in a is to separate adapters from the desired DNA electrophoretically, Nanosep 30K device. The retained material was resuspended and then to recover the DNA from the gel. Because of the in 40 µL dH2O and divided into 10 µL aliquots for analysis. numerous applications that require production of recombinant One aliquot was used as an uncut control while the other DNA molecules, a rapid method is needed for transferring aliquots were diluted into a 20 µL restriction digestion reaction. DNA fragments from a PCR product or other source into the These samples were digested with EcoRI, BamHI, or XbaI desired vector. (Promega, Madison, WI, USA) for 30 min at 37 °C, electrophoresed on a 1.5% agarose gel, and the DNA was Materials and Methods visualized under UV light after staining with ethidium bromide. Optimized DNA Separation and Recovery Rapid Cloning of PCR Fragments Using Ultrafiltration To test the utility of Nanosep devices in practical cloning ® To test the ability of Nanosep ultrafiltration devices to applications, we generated a PCR product from the fern discriminate between PCR products and primers, the recovery Ceratopteris richardii corresponding to a 620 bp conserved characteristics of DNA molecules of two different sizes were region of a phytochrome (PHY) photoreceptor genomic DNA compared for devices containing different MWCO (molecular sequence (3). Genomic template DNA from three 14-day-old weight cutoff) membranes. A 500 µL sample at 100 µg/mL C. richardii gametophyte tissue was isolated by the alkaline (50 µg total) DNA containing either a 50 bp or 500 bp lysis method (7). Forward (5'-AAGGATCCACACGGGAAGTTT- double-stranded DNA fragment was centrifuged at 5,000 x g TGGCC-3') and reverse (5'-TTAAGCTTGATATTCGGGACTCT- in Nanosep devices and recovered in 50 µL TE. Recovered GAAAC-3') primers were constructed with sequences samples were quantitated spectrophotometrically using corresponding to complementary sequences from a genomic absorbance at 260 nm. PHY gene. Restriction enzyme adapter sites for BamHI and The effectiveness of ultrafiltration for PCR product purification HinDIII were engineered two base-pairs downstream from the was examined by observing the recovery of free nucleotides, 5' ends of each primer, respectively, to generate compatible primers, and PCR products following filtration though a selection sticky ends for cloning into the vector polylinker (underlined in of devices containing different MWCO membranes. Before the above sequences). The 25 µL PCR reactions were starting PCR, γ-32P-dATP end-labeled primers as well as 1 µL prepared using PCR Master Mix (Qiagen, Valencia, CA, USA) α-32P-dCTP were added to the PCR reactions. The 400 bp supplemented to 2.5 mM MgCl2, 0.2 µM primers, and 1 µL DNA fragments were synthesized using a custom primer set genomic template DNA. PCR reactions were transferred to a (5'-ACCTATAGCGGTGTGAAATACCGCACAGATGCGTAAG- preheated thermocycler at 90 °C and incubated for 1 min, GAGA-3' and 5'-TGGGCGCTCTTCCGCTTCCTCGCTCACT- then subjected to 35 cycles of 30 sec at 94 °C, 30 sec at GACTCGCTGCGCT-3') corresponding to sequences 45 °C, and 60 sec at 72 °C, followed by a postcycle extension approximately 200 bp upstream and 200 bp downstream for 3 min at 72 °C. A 6 µL aliquot of each reaction was of the pUC18 polylinker. All radioactive PCR products were subjected to eletrophoresis to confirm the size and purity of synthesized at low specific activity by adding 1 µL of labeled the products and to quantitate the fragments for subsequent nucleotide (α-32P-dCTP, 3000 Ci/mmol) to the PCR reaction. cloning. The utility of Nanosep device-based cloning was After synthesis, unincorporated nucleotides were removed by compared with standard fragment cloning methods (5). ultrafiltration (Nanosep 30K device at 5,000 x g for 5 min). The Nanosep device method: To minimize handling time and labor, resulting retentate was recovered in 40 µL TE (10 mM Tris, 1 µg of PCR product and 0.2 µg of pBluescript KS+ vector 1 mM EDTA, pH 7.5) and quantified using gel electrophoresis. (Strategene, La Jolla, CA, USA) were combined and The reaction was run for 30 cycles using standard conditions; simultaneously digested with BamHI and HinDIII directly in a the samples were pooled to ensure consistency, then split into Nanosep 100K device. Digestion was carried out in a total equal aliquots for desalting and concentration in a variety of volume of 50 µL for 2 hr at 37 °C. The digestion products MWCO devices. The retained material was recovered in 20 µL were centrifuged through the Nanosep device at 1,500 x g for TE, electrophoresed using a 10% polyacrylimide Tris-borate gel 5 min, and the retentate was washed twice by addition of 50 µL (BioRad, Hercules, CA, USA), and analyzed by autoradiography. dH2O and centrifugation at 1,500 x g for 5 min. The retained DNA was then resuspended in 8.5 µL sdH2O, 1 µL 10x ligation The retention of biological activity during DNA handling is buffer with ATP, and 0.5 µL ligase. Ligation was allowed to critical. To determine the effect of ultrafiltration on biological proceed directly in the Nanosep device at 17 °C overnight. activity, we synthesized a 400 bp PCR product containing a centrally located polylinker (described