Singular Point Genomic Signature Tags (GST) Protocol for Csp6I- fragments Required reagents and recommended suppliers T4 DNA ligase 350U/?l, equals ~2.8 Weiss U/?l (Takara Biotechnology - 2011A) T4 DNA ligase High Concentration 5 Weiss U/?l (Invitrogen - 15224-041) Csp6I 10U/?l (Fermentas - ER0211) NdeI 10U/?l (New England Biolabs - R0111S) Exonuclease I 20U/?l (New England Biolabs - M0293S) MmeI 2U/?l (New England Biolabs – R0637S) Streptavidin coated Magnasphere Paramagnetic beads (Promega – Z5481) Dynal MPC magnet or other suitable device HPLC purified Oligonucleotides (Invitrogen) 10 x OPA - One Phor All buffer (Amersham, Pharmacia) GlycoBlue 15 mg/ml (Ambion - 9515) Taq DNA polymerase 10x reaction buffer without MgCl2 (Promega - M190) Platinum Taq DNA Polymerase High Fidelity (Invitrogen) MgSO4 solution, 50mM (Invitrogen) Nucleotide mix (dNTP), each at concentration of 10µM (Roche - 1581295) pGEM5 cloning vector (BNL) Calf Intestinal Alkaline Phosphatase (CIP) (New England Biolabs - M0290S) GFX PCR DNA and Gel Band purification kit (Amersham, Pharmacia – 27-9602-01) Acrylamide/Bisacrylamide solution, 30% (Sigma - A3574) Preparation of linker cassettes Name Primer Sequence Csp6I cassette #1, 16S GST TTTGGATTTGCTGGTCGAATTCAACTAGGCTTAATCCGACG Csp6I cassette #2, 16S GST TACGTCGGATTAAGCCTAGTTGAATT Degen.-Csp6I cassette #1, 16S GST P-TTTGTACGGCGGAGACGTCCGCCACTAGTGTCGCAACTGACTA Degen.-Csp6I cassette #2, 16S GST TAGTCAGTTGCGACACTAGTGGCGGACGTCTCCGCCGTACAAANN P=5’-phosphorylated Oligonucleotides for the linker cassettes are dissolved in ddH2O to a concentration of 100 pmoles/?l Pairs are annealed together in the following standard reaction: Csp6I cassette 36 ?l Csp6I cassette#1 oligonucleotide 36 ?l Csp6I cassette#2 oligonucleotide 10 ?l 10xOFA 18 ?l TEsl (super low) (10 mM TrisHCl, pH 8.0; 0.1 mM EDTA-Na3) 100 ?l Degenerated Csp6I cassette 36 ?l Degen.-Csp6I cassette#1 oligonucleotide 36 ?l Degen.-Csp6I cassette#2 oligonucleotide 10 ?l 10xOFA 18 ?l TEsl (10 mM TrisHCl, pH 8.0; 0.1 m M EDTA-Na3) 100 ?l For annealing, the following conditions are used (program in PCR machine): 95?C 2 min 65?C 10 min 37?C 10 min RT 20 min Store at -20?C Optional: check annealing by electrophoresis on a 12% polyacrylamide (30% stock solution) gel, use 20-40 pmol (~ 2 ?l) of each separate strand and annealed product. Preparation of environmental DNA Before a DNA sample is used for the GST protocol it is important that its quality is checked. Critical issues are that the DNA can be digested with Csp6I, and that it is of PCR quality. The best checks are a Csp6I digestion and a PCR reaction using the 26F and 1392R primers on the 16S rRNA gene. The PCR conditions for amplifying the 16S rRNA gene are: 1. 95?C 2 min 2. 95?C 30 sec 3. 52?C 30 sec 35 cycles steps 2-4 4. 72?C 3 min 5. 72?C 8 min 6. 4?C hold 26F primer sequence: AGA GTT TGA TCC TGG CTC AG 1392R primer sequence: ACG GGC GGT GTG TRC An additional option is to run a temperature gradient (48-58?C) in order to determine the optimal temperature for the 16S rRNA gene amplification. The optimum temperature is subsequently used in the LAMP-1 PCR reaction. DNA Digestion DNA is digested with Csp6I. 2-5?g DNA 10?l Fermentas 1x B+ buffer (plus 1xBSA) 2?l Csp61 enzyme (20 units) Final volume 100?l Digest for 2 hrs at 37?C. Heat inactivated the enzyme by incubating the digestion mixture for 20 min at 65?C. The DNA is purified on a GFX column according to manufacturers’ instructions and eluted in 34µl ddH2O. Ligation of Csp6I cassette 5' TTTGGATTTGCTGGTCGAATTCAACTAGGCTTAATCCGACG TTAAGTTGATCCGAATTAGGCTGCAT Remark: the linkers are NOT phosphorilated in order to avoid duplex formation. In addition, the 5’-end of the linker is single stranded. To 34 ?l enzyme digested genomic DNA, add 5.5 ?l 10x T4 DNA ligase buffer (Takara) 13 ?l Csp6I-cassette (~50 fold excess) 3 ?l T4 DNA ligase (Takara) 50 ?l Incubate overnight at 16?C. The ligation product is purified on a GFX column to remove the remaining linker according to manufacturers’ instructions and eluted in 50µl ddH2O. Linear PCR on DNA- Csp6I cassette ligation product (LAMP-1) In this step, a biotinylated primer is used that faces out of the gene of interest. With respect to the 16S rRNA gene, this primer is called 26R and is complementary to the conserved region of base pairs 4-26. The PCR will result in the creation of single stranded, biotinylated DNA fragments that on the 5’ end contain the 26R primer sequence, and on the 3’ end the sequence complementary to the 5’ end of the Csp6I cassette. The following PCR reaction is prepared: To 5 ?l ligation mixture, add: 5 ?l 10x Buffer (Promega, buffer without MgSO4) 2 ?l MgSO4 solution, 50mM 1.5 ?l dNTP mix (10µM solution of each nucleotide) 2 µl 26R-BIO primer (10µM solution): BIO-CTG AGC CAG GAT CAA ACT CT 34.3 µl ddH2O 0.2 ?l Platinum HiFi Taq Polymerase 50 ?l The PCR is carried out according to the LAMP-1 PCR program: 1. 95?C 2 min 2. 95?C 30 sec 3. 52?C 30 sec 35 cycles steps 2-4 4. 72?C 3 min 5. 72?C 8 min 6. 4?C hold Note: the temperature of step 3 might be adapted according to an optimal annealing temperature found when testing the 26F-1392R PCR gradient; the time in step 4 was chosen to allow the synthesis of single stranded DNA fragments with sizes up to 2kb. Synthesis of double stranded DNA fragments. Keep in mind that those ds-DNA fragments that have not been amplified during the LAMP-1 PCR process will be flanked by linkers with a single stranded end. The second primer used in this PCR is directed against the complementary strand of the linker, thus only the ss-DNA fragments that were generated during the LAMP-1 process will be amplified in this second PCR. The following PCR reaction is prepared: To 10 ?l LAMP-1 PCR product, add: 5 ?l 10x Buffer (Promega, buffer without MgSO4) 2 ?l MgSO4 solution, 50mM 1.5 ?l dNTP mix (10µM) 2 µl 26R-BIO primer (10µM) 2 µl Forward primer (10µM): GGATTTGCTGGTCGAATTCAAC 27.3 µl ddH2O 0.2 ?l Platinum HiFi Taq Polymerase 50 ?l The PCR is carried out according to the LAMP-1 PCR program. After the PCR, the product is analyzed on a 0.8% agarose gel and compared to the Csp6I digested DNA. One expects to see amplification of DNA with a maximum intensity at positions between 200-800bp. The amplified fragments will be double stranded and biotinylated. Bind biotinylated fragments to streptavidin beads. Remove 100?l thoroughly resuspended (DO NOT VORTEX) Magnasphere streptavidin beads from the stock into a clean 1.5 ml siliconized or low adhesion (Ambion) microcentrifuge tube and place tube on the magnetic stand. Remove supernatant and wash beads 3x with 400?l 1x B&W (binding and wash) buffer (10 mM TrisHCl, pH 8.0, 2 M NaCl, 1mM EDTA). Resuspend beads in 100?l 1x B&W buffer. Add 50?l 2x B&W buffer to the PCR mixture. Take the total volume of 100?l and add to the suspended beads. Wash the PCR tube with 200 ?l 1xB&W buffer and add also to the beads. Mix gently and incubate at room temperature for 1 hour. DO NOT VORTEX, but make sure beads are fully resuspended. During this incubation the biotinylated fragments will bind to the beads. Flick the solution to mix every 20 minutes. Remove the unbound DNA fragments by washing the beads 3x with 400?l 1xW&B buffer, followed by 2 washings with 400?l TE. Than wash the beads with 100?l restriction Buffer #4 (NEB). Collect beads, carefully remove supernatant and add premixed: 86 ?l ddH2O 10 ?l 10x Buffer #4 1 ?l 100xBSA 1 ?l 4mM SAM (S-adenosylmethionine, comes with MmeI) 2 ?l MmeI (10U/?l) Incubate 1.5 hours 37?C with occasional mixing. Add an additional 2 ?l MmeI and incubate for an additional 1.5 hours 37?C. The GST tags will be released. Collect the beads. The GST tags will be in supernatant. Remove supernatant to clean tube. Rinse beads with 100 ?l TE and combine with 1st supernatant. Add 200 ?l Phenol/Chloroform to sample to remove/inactivate restriction enzyme, recover the supernatant then ethanol precipitate tags: 200 ?l sample 133 ?l 7.5 M NH4OAcetate 3 ?l GlycoBlue 1.0 ml 100% Ethanol Place at -80? C for 1 hour or O/N at -20? C, then spin 30 min at 4? C, wash with 70% EtOH in cold. Resuspend tags in 29.5?l TEsl plus 4?l 10 x T4 DNA ligase buffer (Takara). Degenerate Linker (Csp6I cassette ) ligation This linker contains a Csp6I site preceded by a TTT triplet that serves as punctuation mark to orient the GST tag towards the 16S rRNA gene. Degenerated Linker TTTGTACGGCGGAGACGTCCGCCACTAGTGTCGCAACTGACTA NNAAACATGCCGCCTCTGCAGGCGGTGATCACAGCGTTGACTGAT Ligation reaction: 33.5 ?l MmeI GST products in ligase buffer 3.5 ?l degenerate linker Csp6I cassette @ 10 pmol/?l=35 pmol) Incubate at RT for 15 minutes, then add: 3 ?l T4 DNA Ligase (Takara) 40?l Incubate overnight at 16?C. The final product should be approximately 100bp with two Csp6I sites (GTAC): 5'-TTTGG —30-TCCGACGTACNNNNNNNNNNNNNNNNNTTTGTACGGCGGAGACGTCCGCCACTAGTGTCGCAACTGACTA 3'-AAACC -30-AGGCTGCATGNNNNNNNNNNNNNNNNNAAACATGCCGCCTCTGCAGGCGGTGATCACAGCGTTGACTGAT PCR amplification of GSTs primers: * Biotin-Forward is biotinylated and corresponds to a portion of MmeI linker’s top strand (= GST5 primer): 5'-BIO-GGATTTGCTGGTCGAATTCAAC * Biotin-Reverse is biotinylated and corresponds to a portion of degenerated linker’s bottom strand (= GST6 primer): 5'-BIO-TAGTCAGTTGCGACACTAGTGGC PCR reactions are prepared according to the following table. For a small library, it is sufficient to prepare one PCR reaction of 25 ?l or 50 ?l. Use Promega buffer: do not use a PCR buffer with ammonium sulfate as it is insoluble in EtOH which then causes problems with later steps. 1 x ?l 10 x ?l 20 x ?l Stock Conc. Reagent 18.55 180.5 363 ddH2O 2.5 25 50 10x Promega Buffer 1.0 10.0 20.0 50 mM MgSO4 0.75 7.5 15.0 10 mM each dNTPs 1.0 10.0 20.0 10 ?M biotinyl forward primer (GST5) 1.0 10.0 20.0 10 ?M biotinyl reverse primer (GST6) 0.1 1.0 2.0 Platinum Taq polymerase mix 0.1 5.0 10.0 GST-tag Ligation products 25.0 ?l 250 ?l 500 ?l (five or ten tubes of 50?l) The PCR is carried out according the following protocol (GST PCR). 1. 95?C 2 min 2. 95?C 30 sec 3. 58?C 30 sec 30 cycles steps 2-4 4. 72?C 30 sec 5. 72?C 4 min 6. 4?C hold It might be important to optimize this step. For that, we use different amounts of GST-tag ligation product, e.g. a gradient from 0.1-1.0 ?l in a final volume of 25 ?l. After PCR, the product is checked on a 10% PAA gel (made from a 30% stock solution). Linear amplification to resolve heteroduplexes: LARHD-1 One of the problems of this PCR amplification is the formation of heteroduplexes, this as a result of the homology between the ends of the different tags (due to the linkers that are common for all fragments). A second series of PCR reactions is employed to resolve these heteroduplexes. Depending on the amount of material required, these reactions can be set up at different scales. For a small library it is sufficient to perform the PCR reactions on 10 ?l of the first GST amplification product. The LARHD-1 PCR serves in the first place to generate more PCR material and can eventually be skipped. 1 x ?l 20 x ?l 40 x ?l 50 x ?l Stock Conc Reagent 13.65 273 546 682.5 ddH2O 2.5 50 100 125 10x Promega buffer 1.0 20 40 50 50 mM MgSO4 0.75 15 30 37.5 10 mM each dNTPs 1.0 20 40 50 10 ?M biotinyl forward primer (GST5) 1.0 20 40 50 10 ?M biotinyl reverse primer (GST6) 5.0 100 200 250 1st round amp’d tags 0.1 2 4.0 5 Platinum Taq polymerase mix 25.0 ?l 500?l 1000?l 1250?l (10, 20 or 25 tubes of 50?l) The PCR is carried out according the following protocol (LARHD-1 program, only one cycle) 95?C 2.5 min 58?C 30 sec 72?C 5 min 4?C hold Pool products, saving 10?l for gel analysis (10% PAA gel of 30% stock) if desired. Products should be approximately 100 bp in length. Exonuclease I digestion of primers To the pooled PCR products add E. coli Exonuclease I, 10 Units for 100 ?l of LARHD-1 products. Incubate at 37?C for 60 minutes and then inactivate the Exonuclease I by incubating for 20 minutes at 80?C. Linear amplification to resolve heteroduplexes: LARHD-2 25 cycles of linear amplification with biotinylated forward primer GST5 followed by one cycle of amplification with biotinylated reverse primer GST6. In general a total PCR mix of 500 ?l is prepared which is divided over 5 PCR tubes of 100 ?l. Biotinylated primers are use to later remove the amplified linker sequences using magnetic streptavidin beads. However, an alternative is to purify the GSTs on a preparative PAA gel, in which case there is no need to use biotinylated primers. 1 x ?l 40 x ?l Stk Conc Reagent 17.65 706 ddH2O 2.5 100 10x Promega buffer 1.0 40 50 mM MgSO4 0.75 30 10 mM each dNTPs 1.0 40 10 ?M biotinyl forward primer GST5 1.0 40 1st round amp’d tags 0.1 4.0 Platinum Taq polymerase mix 25.0 ?l 1000 ?l (10 tubes of 100 ?l) The PCR is carried out according the following protocol (LARHD-2, 25-30 cycles) 1. 95?C 2 min 2. 95?C 30 sec 3. 58?C 30 sec 25 cycles steps 2-4 4. 72?C 30 sec 5. 72?C 5 min 6. 4?C hold Subsequently, add 4 ?l biotinylated reverse primer (GST6) and an additional 0.4 ?l of Platinum Taq polymerase to each 100 ?l reaction mix, followed by one cycle of denaturation and extension. The PCR is carried out according the following protocol (LARHD-1 program, only one cycle) 95?C 2.5 min 58?C 30 sec 72?C 5 min 4?C hold Exonuclease I digestion of primers To the pooled PCR products add E. coli Exonuclease I, 10 Units for 100 ?l of LARHD-2 products. Incubate at 37?C for 60 minutes, than inactivate the Exonuclease I by incubating for 20 minutes at 80?C. The LARHD-2 product is purified on a GFX column, this according to the manufacturer’s instructions and eluted in 240 µl ddH2O. Of this sample, 10?l is saved for gel analysis (10% PAA gel of 30% stock). Csp6I digestion to release the GST-tags Digestion is performed at 37?C for 4 hrs. Add to the 230 µl LARHD-2 product: 130 ?l ddH2O 40 ?l Buffer B+ (Plus BSA) from Fermentas 2.0 ?l Csp6I 400 ?l final volume Csp6I (2 ?l) is typically added twice, 2x 2 hr digestions for a total of 4 hrs at 37ºC. Digestion products should be: MmeI linker arm-41mer 1 Biotin-TTTGGATTTG CTGGTCGAAT TCAACTAGGC TTAATCCGAC G AAACCTAAAC GACCAGCTTA AGTTGATCCG AATTAGGCTG CAT plus GST-24mer TACRRRRRRR RRRRRRRRRR TTTG GYYYYYYY YYYYYYYYYY AAACAT plus degenerated linker arm -39mer TACGGCGGAG ACGTCCGCCA CTAGTGTCGC AACTGACTA GCCGCCTC TGCAGGCGGT GATCACAGCG TTGACTGAT-Biotin Phenol/Chloroform (PC) extract on ice, add 40 µl of 3 M NaOAcetate (pH=6) solution, 2.5 ?l GlycoBlue, and 1000 µl 100% ethanol. Chill at -80?C for 1 hour. SPIN IN COLD ROOM (IMPORTANT) Wash pellet in ice-cold 70% Ethanol, and dry. >From this point on you have two alternatives GST recovery protocols are available. The GSTs are either Gel purified, or the biotinylated linkers are removed using streptavidin beads. When streptavidin beads are use, the sample is resuspended in 200 ?l cold TEsl +25 mM NaCl. 1) Elution of TAGS: DO NOT MELT THEM Prepare a 12% Poly acrylamide gel (5.5ml ddH2O, 4.0ml Acrylamide/bis-acrylamide (30% solution, SIGMA #A-3574), 0.5ml 20x TAE, 133µl APS, and 10µl TEMED (SIGMA #T-8133). Run ligation product for 1-1.5 hour at <100volts. Resuspend the above pellet in a volume of ddH2O appropriate to the loading capacity of PAA gel wells minus 10µl. I like to use 15µl ddH20 and add 10µl of liquid streptavidin protein to the resuspended pellet. Leave at room temperature for 10 minutes. During this time pierce bottom of a 0.6ml sterile siliconized tube with the smallest syringe needle possible. Place pierced 0.6ml tube into a 1.5ml Eppendorf tube. Take image of DNA under long wavelength fluorescence. Be sure to clearly see your tags. Cut out band and place it inside the pierced 0.6ml Eppendorf tube and place this tube back inside the 1.5ml Eppendorf tube. Spin at 14,000 rpm for 1 minute. This is done to pulverize the acrylamide gel so at to best be able to draw our tags from it. If there is any unpulverized gel remaining inside the 0.6ml tube remove it with a pipette tip and place it with the pulverized portion in the 1.5ml Eppendorf tube. Discard 0.6ml tube when this is completed. Then to each tube add: 250µl TEsl 50µl 7.5M Ammonium Acetate Add mixture to cover crushed gel slice and incubate at 37ºC in an air incubator for 1 hr to ON with occasional mixing (I would advise 5 hours, and disadvise leaving it overnight). The tags will diffuse out of the gel, then to collect them place contents of tube over a filter column (Millipore Ultrafree-MC, 30000 NMWL filter unit, N9JKK1530) to remove acrylamide and spin at 5000rpm for 2 minutes. Remove the flow-through and place into another tube. Take 50µl of TEsl and place it in the 1.5ml Eppendorf tube that contained your crushed PAA gel (this is done to wash out the remaining traces of PAA and tags), place this over the same filter column and again spin at 500rpm for 2 minutes. Pool flow-through and then do an EtOH precipitation. 300µl Sample 2.5µl GlycoBlue 1240µl EtOH If you need to reprecipitate: dissolve sample in COLD TEsl plus 0.3M Na Acetate, pH 6.0, use 2.5x vol EtOH. Spin and dissolve sample in TEsl PLUS 25mM NaCl – KEEP COLD. OR 2) Bind biotinylated arms to Dynal streptavidin beads: Use 200 ?l Promega beads, three times washed in 1x B&W. Washed beads are resuspended in 200 ?l 2x B&W, add Csp6I digest, mix gently at room temperature for 15-30 min. Collect the beads and recover the supernatant, which will contain the unbound fraction --THESE ARE THE GSTs. Wash the beads with 100 ?l 1x B&W buffer, pool with the first fraction and precipitate the GSTs with 2.5 vol. ethanol at -80?C (incubate 1 or 2 hours). SPIN IN COLD ROOM (IMPORTANT) Resuspend in 200 ?l 0.3 M Na acetate, pH6.0, and add 2.5 vol. (500 ?l) Ethanol. Place at 80?C for 1 to 2 hours, spin at 4ºC, wash pellet in ice-cold 70% Ethanol, and dry. This second precipitation-wash step is necessary to remove the high salt. Self-ligation of GST tags to form concatemers Resuspend GST pellet in 12.5?l TEsl on ice, add 1.5?l 10 x T4 DNA ligase buffer (Takara) 1.0?l T4 DNA Ligase (Invitrogen-High Conc) Incubate overnight at 16?C Add 25 ?l TEsl+25mM NaCl, heat for 2.5 min at 65?C, quench on ice, add 4?l 80% glycerol, mix and apply to a single slot of an 8 slot 0.75% Low Melt agarose minigel bracketed by 100 and 500 bp ladders. Electrophorese and cut out concatemers. Note in some cases we cut the gel to remove tags of < 100-250 bp, and reversed the gel’s polarity to concentrate the DNA prior to elution. Tags are purified using GFX Spin columns (Amersham, Pharmacia). Elute with 180 ?l ddH2O add 20 ?l 3M NaOAc, pH 6.0, 2 ?l GlycoBlue 500?l EtOH, chill spin, wash with 70% EtOH, dry, up in 8 ?l TEsl plus 1 ?l 10x T4 DNA ligase buffer Takara, mix, add 0.5 ?l NdeI cut pGEM5, heat at 65?C for 30 sec, quench on ice, add 1 ?l T4 DNA ligase (Takara), mix and incubate at 16?C for several hrs (at least 2h). Dilute to 50 ?l by adding 40 ?l 1X T4 Ligation buffer plus 1 ?l T4 DNA ligase, incubate O/N at 16?C, PC extract, EtOH ppt from NaOAc + GlycoBlue Resuspend sample in 10 -15 ?l ddH2O, Electroporate 5 ?l sample into 50 ?l TOP10 competent cells. Phenotypically express each sample in 1 ml 2xYT for 1 hr at 37?C with shaking, pool = 3 ml. Plate 200, 100, 50 and 25 ?l onto prewarmed 2xYT plates + 50 ?g/ml kanamycin. Incubate plates overnight. A good library should provide 100-200 colonies on the 25 ?l to 50 ?l platings. Polyacrylamide gel electrophoresis The desired polyacrylamide mix is prepared according to the following table: 7.5% 10% 12.5% Acryl/Bisacrylamide 2.5 3.3 4.0 20x TAE buffer 0.5 0.5 0.5 ddH2O 7.0 6.2 5.5 The mixture is degassed, and than 133 ?l 10% APS and 8 ?l TEMED (Sigma – T8133) are added. The gel is run at 15m Amp.