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Protocol For: SPLICE: A Technique for Generating In Vitro Spliced Coding Sequences from Genomic DNA
Sponsored,vendor-submitted protocol    Published in November 2009 (p.67) DOI: 10.2144/000113039

Materials Reagents

Source of Genomic DNA (e.g., liver)

Tris Base (Cat# T1503-1KG; Sigma-Aldrich, Gillingham, UK)

Ethylenediaminetetraacetic Acid (EDTA; Cat# 100935V; VWR, Lutterworth, UK)

Sodium Dodecyl Sulphate (SDS; Cat# L-4509; Sigma-Aldrich)

Proteinase K (Cat# P2308-25MG; Sigma-Aldrich)

Ethanol [70%; take 70 ml absolute ethanol (Cat# 1010774Y; VWR) and add 30 ml of sterile water]

Acetone (Cat# 100035R; VWR)

N,N-Dimethylformamide (DMF; Cat# D4551-500ML; Sigma-Aldrich)

KOD Hot Start DNA Polymerase Kit, with 200 U KOD hot start DNA polymerase enzyme, 10× PCR buffer for KOD hot start DNA polymerase, 2 mM dNTP mix and 25 mM MgSO4 (Cat# 71086-3; Novagen, Nottingham, UK) or Platinum Taq DNA Polymerase Kit, with Platinum Taq DNA polymerase enzyme, 10× PCR buffer for Platinum Taq DNA polymerase and 50 mM MgCl2 (Cat# 10966-018; Invitrogen, Paisley, UK)

Distilled and Deionized Water, autoclaved, to be used for all solutions and dilutions

Synthetic Oligonucleotides, 100 µM stock solution (Sigma-Genosys, Haverhill, UK)

HyperLadder II DNA Marker (Cat# 33039; Bioline, London, UK)

Bromophenol Blue (Cat# B/P620/44 59; Sigma-Aldrich)

Xylene Cyanol (Cat# X-4126; Sigma-Aldrich)

Agarose (Cat# BIO-41025; Bioline)

Tryptone (Cat# VM423731; Merck, Nottingham, UK)

Agar (Cat# 20-767.298; VWR)

Yeast Extract (Cat# L21; Oxoid, Basingstoke, UK)

Sucrose (Cat# 102747E; VWR)

Urea (Cat# 443876Y; VWR)

NaCl (Cat# S-3160-65; Fisher Scientific, Loughborough, UK)

50× Tris-Acetate-EDTA (TAE) Buffer (Cat# EC-872; National Diagnostics, Hessle, UK)

Ethidium Bromide Solution (EtBr; Cat# 46067; Fluka Biochemika, Gillingham, UK)

Gel Extraction Kit (Cat# 28706; Qiagen, Crawley, UK)

T4 DNA Ligase (Cat# M1801; Promega, Southampton, UK)

Ampicillin Sodium Salt (Cat# 171254; Calbiochem, Nottingham, UK)

EcoRI (Cat# R6011; Promega) and SalI Restriction Enzymes (Cat# R6051; Promega)

Cloning Vectors [e.g., pBluescript II KS+ (Cat# 212207; Stratagene, Amsterdam, The Netherlands) or Expression Vectors (e.g. pMT4; a derivative of pMT2 expression vector with an additional bovine 1D4 epitope (Reference 1)]

JM109 Competent Cells [Genotype: endA1, recA1, gyrA96, thi, hsdR17 (rk-, mk + ), relA1, supE44, Δ(lac-proAB), (F′, traD36, proAB, laqIqZΔM15); Cat# L2001; Promega]

GenElute Plasmid Miniprep Kit (Cat# PLN350-1KT; Sigma-Aldrich)

BigDye Terminator Version 3.1 Cycle-sequencing Kit (Cat# 4337455; Applied Biosystems, Warrington, UK)

Equipment

0.2 ml Thin-wall PCR Tubes (Cat# TA511; Appleton Woods, Birmingham, UK)

1.5 ml Eppendorf Tubes (any suitable company)

Touchgene PCR Machine (Techne, Fradley, UK)

Gel Electrophoresis Equipment (Sub-Cell Model 192; Biorad, Hemel Hempstead, UK)

3730 DNA Analyzer and Sequencer (Applied Biosystems)

Filter Tips (various volume sizes; Starlabs, Milton Keynes, UK)

Protocol Determine the Genomic Structure for the Gene of Interest to Be Amplified

1. Establish the exon-intron and intron-exon boundaries for each exon to be amplified. Where the genome of a particular species of interest has been sequenced, gene annotation, including knowledge of exonintron and intron-exon boundaries, may have been completed. Where this is not the case, comparison by using a pairwise alignment algorithm between a previously sequenced cDNA species and the genomic sequence covering the gene locus of interest will allow for manual identification of intronic regions to be removed by applying the SPLICE technique outlined in this study. Care should be taken to ensure that all putative donor and acceptor splice sites satisfy the GT-AG rule (1) to ensure the correct positioning for subsequent primer design. For this purpose, interrogation of the many expressed sequence tag (EST) databases that now exist, by applying the basic local alignment search tool (nucleotides) algorithm, blastn, may serve as a source of obtaining RNA/cDNA sequence information. Conversely, if cDNA sequence but not genomic DNA (gDNA) information is readily available, comparison of known exon-exon boundaries within a particular cDNA sequence with sequence obtained from other species may be sufficient since exon-intron and intron-exon boundaries are highly conserved throughout evolution. For an increasing number of genes, alternative splicing of internal exons or differential usage of flanking exons may result in different isoforms of a particular transcript that exhibit tissue-specific expression and a wide variety of functions (2,3,4,5,6,7). In these cases, knowledge of both transcript sequence and genomic structure is a prerequisite to further analysis. Nonetheless, where alternative splicing is known, the SPLICE technique may be used to generate any splice variant required for in vitro experimentation. Applied more universally, the SPLICE procedure may be used to fuse, insert, or delete any region(s) of DNA from a target sequence.

Primer Design for SPLICE Assay

2. Design a set of primers to encompass each exon to be amplified similar to that outlined in Figures 1 and 2 of the original article (see www.BioTechniques.com/article/112588). All internal primers should be 30 nucleotides in length to ensure specific binding to the target region of gDNA and encompass an exon-exon boundary or a fusion point between the two DNA sequences to be united. For forward internal primers, the first half of the oligolucleotide sequence should correspond to the last 15 nucleotides of an upstream exon, with the second half being identical to the first 15 nucleotides of a downstream exon. The reverse internal primers should comprise the reverse complement sequence of the forward primer. In all cases, the fusion point between the two DNA sequences being joined will be located at the center of each internal oligonucleotide. Depending on the requirements of the downstream assay, modifications may be included in some of the primers at this design stage, such as the inclusion of restriction enzyme recognition sites in the two outer (sense and antisense) primers for ease of cloning, the addition of a Kozak consensus sequence upstream of the translation initiation codon in the outer forward primer to ensure efficient protein production in a downstream assay, and replacement of the translation termination codon with partial linker sequence that is inframe with a downstream reporter gene of interest.

Generally, the use of sophisticated and expensive software is not required when designing SPLICE primers. In the majority of experiments, overlapping primers will be identical to the target sequence. However, in some cases where mismatches are required (e.g., for mutagenesis), it is highly recommended that any nucleotide differences between the primer and target sequences are restricted to the central 10 nucleotides of each primer to allow for efficient binding of primer to target sequence, as well as the annealing of overlapping PCR products during the fusion stages of the SPLICE procedure. Where mismatches from the target sequence are to be tolerated, it is imperative that each overlapping sense and antisense primer contains a complementary nucleotide. In all cases, we recommend that oligonucleotides should be at least 30 nucleotides in length with one half of the primer designed to the 3′ end of an upstream exon and the other half of the primer to the 5′ end of a downstream exon. Due to limitations in the desired template sequence to be amplified, it is not always possible to design primers that fulfill the usual criteria for primer design of a GC-clamp with 50% GC content. However, the robustness of our method should permit successful amplification with primers that end with any nucleotide and possess GC contents between 20% and 80%. Should some primers be designed to areas that are GC-rich and are thus prone to inhibitory secondary structures, rigorous PCR optimization supplemented with PCR additives such as DMSO or formamide to negate these inhibitory effects may be required.

Genomic DNA Extraction

3. Extract gDNA of sufficient quality to allow amplification. Genomic DNA from any source should be sufficient since the majority of exons are small enough to be effectively amplified from even partially degraded gDNA. To this effect, even ancient DNA may be used as template for the SPLICE assay. In this study, gDNA was extracted from the brown lemur liver and kindly donated by Professor John Mollon (University of Cambridge, UK). Many methods of gDNA purification exist based on phenol/chloroform extraction, silica-column technologies, and direct precipitation. However, we have found the following rapid protocol suitable for generating PCR-quality gDNA from liver tissue.

4. To a small piece of liver (or other tissue) add 0.5 ml of extraction buffer (10 mM Tris base, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% (w/vol) SDS).

5. After gentle mixing, add 20 µl Proteinase K (10 mg/ml), mix and incubate overnight at 55°C.

6. Centrifuge at 13,000 rpm for 10 min and transfer the supernatant to a fresh 1.5 ml eppendorf tube.

7. Add 1 ml of 70% (v/v) acetone/5% (v/v) dimethylformamide (for 5 ml: 3.5 ml acetone; 250 µl DMF; 1.25 ml sterile water).

8. Centrifuge at 13,000 rpm for 10 min and remove the supernatant.

9. Wash the pellet with 1 ml of 70% ethanol, centrifuge at 13,000 rpm for 2 min, and remove the ethanol supernatant.

10. Slightly dry and resuspend the DNA pellet in 100 µl TE buffer (10 mM Tris base, pH 7.5; 1 mM EDTA, pH 8.0), using a 1 in 10 dilution in subsequent PCR experiments. It is important not to overdry the pellet as it may be difficult to resuspend the gDNA.

11. Run a small sample (∼5 µl) of the extracted genomic DNA, supplemented with 1× loading buffer (make a 5× stock containing 4 M urea; 50% (w/vol) sucrose; 50 mM EDTA, pH 7.0; a few grains of bromophenol blue and xylene cyanol), on a 0.5–1% (w/vol) agarose/EtBr gel to assess gDNA quality and viability in subsequent PCR amplifications. Apply a voltage of 100 V and electrophorese for 30–60 min. Take a photograph of the electrophoresed gel.

First-Round SPLICE Amplification of Discrete Exonic Regions

12. Dilute all oligonucleotides to 10 µM with sterile water, mix to homogeneity, and place on ice. Thaw all PCR reagents (10× KOD DNA polymerase buffer, 2 mM dNTP mix, 25 mM MgSO4 solution), mix to homogeneity and place on ice.

13. To each thin-wall PCR reaction tube* add the components listed in Table 1.





14. To the above mixture, add 1.5 µl of each sense and antisense primer (final concentration of 0.3 µM) designed to specifically amplify each discrete exonic region.

15. Add 1 µl of gDNA template (10 ng to 100 ng), mix the contents gently, and spin briefly to collect the PCR mixture at the bottom of the tube. It is recommenced that a no-template control is included to detect the presence of contamination.

16. Perform the PCR under the conditions listed in Table 2.





Visualization and Purification of First-Round Products

17. Run 20% (10 µl) of the PCR products from each tube, supplemented with 1× loading buffer, on a 1–2% (w/vol) agarose/EtBr gel. Include a DNA marker (500 ng; HyperLadder II DNA marker, Bioline) to determine the size of each product. Apply a voltage of 100 V and electrophorese for 30–60 min. Take a photograph of the electrophoresed gel.

18. For each reaction, a single distinct product of the expected size should be observed. If this is the case, excise each band with a clean blade and place into a fresh 1.5 ml tube.

19. Weigh each gel fragment and purify using the Gel Extraction kit (Sigma-Aldrich) by following the manufacturer's instructions. Elute in 30 µl elution buffer.

Second-Round SPLICE Amplification to Fuse First-Round PCR Products

20. From the gel photograph, estimate the concentration of each purified product by comparing each PCR product with the quantitative DNA marker.

21. Determine the equimolar concentration of each purified first-round PCR product to be combined in subsequent rounds of amplification. Routinely, we found that the total volume of input template (collectively consisting of the total number of first-round PCR products to be fused together) required for the second amplification step was rarely greater than 10 µl.

22. Set up the second-round amplification in a similar way to that used for the first-round PCR protocol (steps 12–16). Once again, thaw all PCR reagents (10× KOD DNA polymerase buffer, 2.5 mM dNTP mix, 25 mM MgSO4 solution), mix to homogeneity and place on ice.

23. To each thin-wall PCR reaction tube* add the components listed in Table 3.





24. To the above mixture, add 1.5 µl of each sense and antisense primer (final concentration of 0.3 µM). It is important that only the two outermost primers are used in this reaction to allow for the SPLICE technique to work efficiently and allow the desired single continuous DNA fragment to be enriched in the product population.

25. Add the equimolar combination of purified first-round PCR products as calculated in step 21. If the volume of the second-round template is less than 10 µl, add sterile water up to this total volume. If the template volume is greater than 10 µl, the total amount of water added to each PCR mix during step 23 should be decreased accordingly to maintain a total PCR volume of 50 µl. Gently mix the contents and spin briefly to collect the PCR mixture at the bottom of the tube. To assess the potential carryover of contamination, second-round reactions may be included that use the first-round no-template products in the same proportions as those used in reactions containing purified first-round PCR products. As with the first-round PCR protocol, separate no-template controls should be included.

26. Perform the PCR under identical conditions to those used for the first-round amplification reaction as outlined in step 16.

Visualization and Purification of Second-Round Products

27. As before, run 20% (10 µl) of the PCR products on a 1–2% (w/vol) agarose/EtBr gel and subject to electrophoresis for 30–60 min. Include a DNA marker (500 ng; HyperLadder II DNA marker, Bioline) to determine the size of each product. Take a photograph of the electrophoresed gel prior to excising each distinct band with a clean blade. Place each gel fragment into a fresh 1.5 ml tube, weight, and purify using the Gel Extraction kit (Sigma-Aldrich) by following the manufacturer's instructions. Elute in 30 µl elution buffer.

Cloning of SPLICE Products and Sequencing

28. Once purified, the final product may be directly sequenced using a BigDye Terminator version 3.1 cycle-sequencing kit as outlined in the manufacturer's instructions. Alternatively, the purified PCR product may be blunt-end ligated to a suitable vector or digested directly with appropriate restriction enzymes and subcloned as detailed in Table 1. It is imperative that all amplified DNA fragments are sequenced in both directions to ensure that any sequence errors are absent in the final product. In our study, full-length products were gel-purified, digested with EcoRI and SalI, subjected to gel electrophoresis, and gel extracted to remove all unwanted reagents. Purified fragments were ligated into a pMT4 expression vector (8) by using T4 DNA ligase, transformed into JM109 competent cells and grown on LB-ampicillin-agar [1% (w/vol) tryptone, 0.5% (w/vol) yeast extract, 1% (w/vol) NaCl, 100 µg/ml ampicillin, supplemented with 1.5% (w/vol) agar] plates using standard procedures. All colonies were picked and used to inoculate LB broth supplemented with ampicillin and grown overnight (∼12–16 h) at 37°C. Plasmid DNA was purified by a GenElute Plasmid Miniprep kit according to manufacturer's instructions and subjected to sequence analysis. Troubleshooting guidlines for many of the steps involved in the SPLICE protocol are listed in Table 4





Correspondence

Address correspondence to Wayne L. Davies at his present address: Nuffield Laboratory of Opthalmology, University of Oxford, Level 5-6 West Wing, The John Radcliffe Hospital, Headley Way, Oxford, OX3 9DU, UK. e-mail: [email protected]

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