RESOLVING DNA AMPLIFICATION PRODUCTS
Introduction
Materials
Electrophoresis
Silver staining
Methods
DNA separation
Silver staining
Reagent handling
Comments
Resolving DAF fingerprints
Silver staining
Trouble-shooting
Polyester film alignment and application
Gel casting
Gel loading
Electrophoresis
Gel handling and staining artifacts
RECOVERING AMPLIFIED DNA
Introduction
Materials
Methods
Comments
Number of recovery cycles
Factors affecting recovery
Applications
RESOLVING DNA AMPLIFICATION PRODUCTS
Introduction
The scanning of nucleic acids by amplification produces an array of DNA products that can be resolved using a variety of methods. As originally described, DAF and AP-PCR use PAGE, while RAPD analysis separates amplification products by electrophoresis in agarose gels. These techniques differ also in the way how nucleic acid fragments are detected; DAF uses silver staining, AP-PCR uses autoradiography, and RAPD uses staining with ethidium bromide. These procedures provide simple, fast and low cost analysis of amplification products. However, many researchers would prefer avoiding the use of radioactivity, acrylamide, and ethidium bromide. Despite its low resolving power, the familiarity and simplicity of agarose gel electrophoresis has made RAPD popular. However, it is a poor strategy for the analysis of complex fingerprints. Alternatively, PAGE has traditionally offered a superior separation of nucleic acids, proteins and polysaccharides. Acrylamide polymerization onto backing supports such as polyester films or glass has simplified gel handling and improved the overall performance of the technique. In particular, the coupling of PAGE and optimized silver staining protocols that detect nucleic acids at the picogram level provides the speed, simplicity and high resolution needed in nucleic acid scanning applications (Bassam et al. 1991, Bassam and Caetano-Anollés 1993, Caetano-Anollés and Bassam 1993, Caetano-Anollés and Gresshoff 1994a, Bassam and Bentley 1995). This section focuses on several polyacrylamide-based separation techniques that can be used to separate DAF or differentially displayed RNA profiles. It also describes the staining of nucleic acid fragments with silver. Return
Materials
Electrophoresis
1. Tris-borate-EDTA (TBE) buffer stock (10x): 1M Tris-HCl, 0.83M boric acid, and 10 mM sodium EDTA (pH 8.3).
2. Acrylamide-crosslinker-urea stock solution (1x): 9.8% acrylamide, 0.2% piperazine diacrylamide, and 10% urea in 1xTBE.
3. Ammonium persulfate (10% stock).
4. N,N,N',N'-tetramethylethylenediamine (TEMED).
5. Loading buffer: Formulation A: 30% urea, 0.08 % xylene cyanol FF (Caetano-Anollés et al. 1991); Formulation B: 40% urea, 3% Ficoll, 0.02% xylene cyanol, 0.02% bromophenol blue, in 1xTBE (Bassam and Bentley 1995).
6. Electrophoresis materials: Polyester gel-backing film (GelBond PAG, FMC Bioproducts, Rockland, ME; or other silanized plastic sheets), membrane syringe filters, and electrophoresis apparatus.
Silver staining
1. Fixative/stop solution: 7.5% [v/v] glacial acetic acid.
2. Silver impregnating solution: 1g/l silver nitrate, 1.5 ml/l 37% formaldehyde.
3. Developer: 30 g/l sodium carbonate, 3 ml/l formaldehyde, 2 mg/l sodium thiosulfate.
4. Staining equipment: Staining trays with flat bottoms and straight sides (if desired, use clear plastic lids from 1 ml pipette-tip racks), and shaker. Return
Methods
DNA separation
Amplification products can be separated in vertical (Caetano-Anollés and Bassam 1993) or open-faced polyacrylamide slab gels (Allen et al. 1989, Doktycz 1993), and by using a semi-automated miniaturized electrophoretic and staining device (PhastSystem, Pharmacia LKB, Piscataway, NJ). I will describe DNA separation by vertical denaturing PAGE using 0.45 mm thick polyacrylamide gels (8x10 cm) backed on polyester film.
1. Assemble each electrophoretic rig under running distilled water to avoid dust particles. Place the large glass plate on clamp assembly, then the polyester backing sheet (hydrophilic side up) in tight apposition to the large glass plate, spacers, and finally the small glass plate. Make sure all rig components are flush against the bottom before tightening assembly screws.
2. Dry the gel rig in a dust-free area in the dark (backing sheets are light-sensitive), overnight. If desired, use an oven at 40-50C to speed the process. Alternatively, subject the gel rigs to a 85% ethanol rinse before drying.
3. Assemble gel rigs in casting stand.
4. Prepare to cast 4-15%T and 2%C polyacrylamide-urea gels. Mix 10 ml of the acrylamide-crosslinker-urea stock solution with 150 µl ammonium persulfate and 15 µl TEMED.
5. Deliver the gel mix by injection through a 0.45 µm-pore size membrane and insert Teflon comb. The gel mix begins to set in about 2 min and should fully polymerize in 30 min.
6. Attach gel rigs to electrode core, place into buffer tank, and fill buffer reservoirs with 1xTBE.
7. Remove combs, rinse well with buffer using a fine-gauge syringe needle, and pre-electrophorese gels at 150V for at least 5 min.
8. Rinse and then load wells with 3 µl of a dilution of each amplification reaction (usually containing 30-40 ng DNA) mixed with 3 µl of loading buffer.
9. Electrophorese at 150 V for about 60-90 min.
10. Disassemble gel rigs and remove backed gels
Silver staining
The polyester-backed gels can be fixed and stained with silver using the procedure of Bassam et al. (1991). The stain detects about 1 pg DNA per squared mm band cross-section, and is commercially available (Promega Corp., Madison, WI). During steps 2-6 of the following protocol subject gels to agitation on a shaker at 50 rpm.
1. Place gels in staining trays.
2. Fix the gels in fixative (acetic acid solution) for 10 min.
3. Wash the gels 3 times with distilled water, 2 min each time.
4. Impregnate with silver solution for 20 min.
5. Rinse with distilled water for 5-20 s.
6. Develop the image with developer solution kept at 8C. Optimum image contrast and minimum background is generally attained in 4 min.
7. Stop image development in stop (acetic acid) solution for at least 1 min. Wash extensively with water.
8. Dry stained gels at room temperature and store in photographic albums.
Reagent handling
Reagents used in DNA separation must be electrophoresis grade and those used in silver staining of high purity analytical grade. Acrylamide solutions are light sensitive and should be stored in the dark at 4C. For silver staining, use relatively fresh formaldehyde and never store it in the cold. Prepare impregnation and developer solutions in advance and store at room temperature. Add formaldehyde and freshly prepared (at least weekly) sodium thiosulfate prior to staining.
Acrylamide is a potent neurotoxin and silver is toxic. Handle these solutions with care and dispose of them appropriately. Recycle the used silver by precipitation with NaCl. Return
Comments
Resolving DAF fingerprints
DAF amplification products can be electrophoretically separated in polyester-backed polyacrylamide gels. DNA separation using vertical, open-faced discontinuous (isotachophoresis) or miniaturized electrophoretic units produces reliable and clearly resolved fingerprints after silver staining (Fig. 2). Polyester backing films constitute an improved support for polyacrylamide gels. These films bind covalently to the acrylamide monomers during polymerization and provide better handling, staining and preservation of the gels. If these films are unavailable, the gels can be attached to glass or plastic via a silane bridge using bind-silane (gamma-Methacryloxy-propyl-trimethoxysilane) solutions.
A) Polyacrylamide concentration
A wide range of gel configurations are possible. Pore size in polyacrylamide gels can be increased by either diluting total monomer components [ie. decreasing polyacrylamide concentration (%T)] or by increasing the percent of cross-linker (%C). At a fixed %T, porosity also increases with both higher and lower %C levels following a nearly parabolic function with a minimum at about 5%C. We have routinely resolved DAF patterns using 4%T:5%C polyacrylamide gels (Caetano-Anollés and Bassam 1993). However, lower crosslinker levels permit the use of higher %T gels without the risk of cracking or peeling away from the backed gels and appear to increase the quality of the silver stain (Bassam and Bentley 1995). An optimal formulation for the analysis of DAF products in the 100-700 bp range is 10%T:2%C. Lower polyacrylamide concentrations can be used when a higher range of products needs to be examined, for example when using native Thermus aquaticus polymerases. When using non-backed gels, the more classical 4-5%T:3-5%C formulations are better suited, as they provide better handling qualities. Mobility modifiers such as glycerol can also be used to tailor DNA separation. Figure 2 shows the effect of 5% glycerol on fingerprint distribution along the gels.
Denaturing and nondenaturing PAGE can be used. However, nondenaturing gels not only separate fragments according to their molecular weight but also according to nucleic acid sequence and base composition. Inclusion of passive denaturants such as urea or formamide in the polyacrylamide gel suppresses base pairing and eliminates the influence of base composition. The presence of denaturants enhance band resolution.
B) Sample loading
Difficulties perceived during PAGE have been classically associated with gel casting, gel handling, and sample loading. While polymerization onto backing supports has simplified the casting and handling of the polyacrylamide gels, sample loading still requires of the delicate hand and somewhat artisanal nature of the researcher. However, the efficacy of loading depends on selecting a suitable loading buffer and on simplifying experimental manipulations. Loading buffers should contain the sample within the well or the gel surface, undiluted, until DNA fragments have entered the polyacrylamide matrix. Moreover, these buffers should not interfere with normal electrophoresis of the samples. When using denaturing urea-polyacrylamide gels avoid sucrose or glycine containing buffers. Urea, Ficoll, formamide, and even glycerol can be used if added in suitable concentrations. Samples can be applied with the help of small-bore flat pipette tips by deposition on the bottom of the well. Alternatively, normal pipette tips (for 20-µl or 200-µl pipettes) can be used. In this case, the wells should be casted only about 5 mm deep and the pipette tip should be placed vertical immediately on top of the well before sample "dropping". The two alternative loading procedures produce identical results.
C) PhastSystem
Nucleic acid scanning techniques have the potential of analyzing thousands of individuals in plant and animal breeding applications. While DNA extraction remains the limiting step, automated workstations that use pipetting robots to assemble amplification reactions (Harrison et al. 1993, Garner et al. 1993) and real time DNA separation of amplification products using fluorophores or capillary electrophoresis (CE) promise to speed pre- and post-amplification steps. Currently, there is a need for a relatively inexpensive DNA separation alternative that would still fulfill high throughput analysis. Semi-automatic separation and staining of DAF fragments in miniature polyacrylamide gels using the PhastSystem can resolve DNA fingerprints with high throughput and simplify detection of diagnostic DNA polymorphisms. The procedure combines our ultrasensitive silver staining technique with vitually complete automation of sample loading, electrophoresis and staining (Baum et al. 1994, G. Caetano-Anollés and B.J. Bassam, unpublished), allowing to process over 180 samples/day per unit with minimal manipulation.
Although requiring the added expense of proprietary reagents and consumables (US$ 8/gel), the PhastSystem eliminates gel preparation (with the use of pre-cast gels polymerized onto clear backing film), permits fast electrophoretic performance (30 samples/2 gels/30 min using one unit), high throughput (30-60 samples/h vs. 15 samples/h using vertical PAGE), and reproducible and sensitive semi-automated DNA separation and staining due to pre-programmed conditions).
Mobility shifts vary with gel concentration and the use of homogeneous or gradient gels. Good separation of high molecular weight fragments was observed with 12.5% and 20% homogeneous, and 8-25% gradient polyacrylamide gels. Separation of 200-1000 bp fragments was optimal when using 10-15% polyacrylamide gels. Overall, DNA distributes differently than in vertical PAGE, offering superior resolution of higher molecular weight DNA fragments and allowing identification of valuable DNA polymorphisms unresolved using more traditional gels.
The discontinuous buffer system resolves DNA poorly unless a long pre-run of 100 Vh generates an appropriate in-gel ion concentration following the buffer front. The existence of a stacking gel, a uncommon practice in DNA separation, appears advantageous. However, Phastgels have a restricted resolving range (about 20 mm), which together with loading constraints result in the general loss of resolution. In particular, fragments smaller than 100-150 bp are poorly resolved due to gel compression and difussion of the buffer front. The stained plastic-backed gels are small, making analysis difficult without magnification, and are delicate, requiring utmost care during handling. Despite these limitations, amplification products are well visualized and can still be recovered succesfully from these miniature discontinuous plastic-backed matrices using the procedure of Weaver et al. (1994).
D) Alternative separation techniques
Fluorophore-labelled DAF products can be sized by laser scanning with automated DNA sequencers (Caetano-Anollés et al. 1992a), by capillary electrophoresis (CE) (Caetano-Anollés et al. 1995), or mass spectrometry (M. Doktycz, pers. commun.). Typical DAF profiles can be produced by CE in about 30 min. Some of these real-time analysis techniques increase resolution but lack throughput, and are limited by availability of proper instrumentation. Recently, other approaches that rely on the separation of single-stranded DNA in polyacrylamide gels according to size and base composition have been used. Denaturing gradient gel electrophoresis (DGGE) (He et al. 1992, Dweikat et al. 1993), temperature sweep gel electrophoresis (TSGE) (Penner and Bezte 1994), and analysis of single strand conformation polymorphisms (SSCPs) (McClelland et al. 1994), appear to increase resolution and detection of polymorphic DNA.
An emerging strategy able to probe DNA sequence without resorting to any separation of DNA is the use of oligonucleotide arrays (Chetverin and Kramer 1994, Beattie et al. 1995). The technique is based on solid phase oligonucleotide synthesis and nucleic acid hybridization. Arrays of short oligonucleotides immobilized on solid supports can be used to survey by hybridization the sequence of amplification products generated by nucleic acid scanning. This strategy, nucleic acid scanning by hybridization (NASBH) can detect single nucleotide polymorphisms through multiple pairwise comparisons (Salazar and Caetano-Anollés 1996).
Silver staining
Silver staining detects nucleic acids with high sensitivity, avoiding fluorophore or radioisotopic labeling. Silver staining of complex nucleic acid profiles separated in polyacrylamide gels provides high band resolution but can be cumbersome and difficult to reproduce if an adequate technique is not used. A simple acidic silver stain that detects picogram quantities of nucleic acids separated on polyester-backed polyacrylamide gels (Bassam et al. 1991) has been developed recently to provide unsurpassed sensitivity and reproducibility. The technique has few steps and reagents, is fast, and produces the least number of staining artifacts (Bassam and Caetano-Anollés 1993; Caetano-Anollés and Gresshoff 1994a), and has even been used in DNA sequencing applications (Storts et al. 1993). The silver stained polyester-backed gels can be preserved for many years by drying, and act as safe repositories of electrophoresed DNA amplification products. These gels permit the retrospective recovery and examination of nucleic acids, constitute experimental records, avoid the costs of photography, and allow easy densitometrical scanning of DNA profiles for data analysis. A desired band, whether part of a simple or complex array of DNA fragments in a profile, can be directly excised and used as template for further amplification. For a detailed description of band recovery from silver stained gels see section
The silver staining protocol is based on a photochemically-derived silver stain originally designed for the staining of proteins (Goldman and Merril 1982). The technique is based on the use of silver nitrate as the impregnating agent and formaldehyde in an alkaline environment as the reducer. Impregnation is done with relatively low concentrations of silver in the presence of formaldehyde. Image development occurs by reduction of silver to metallic silver by formaldehyde at low temperature (8-12C) and alkaline pH. The metallic silver deposits in the immediate vicinity of the staining substratum while the complexant sodium thiosulfate keeps silver reduction in the polyacrylamide matrix to a minimum. Silver complexation alters the kinetics of silver reduction and helps minimize background staining. Image development is finally stopped by decreasing pH, preferably with the use of weak acids such as acetic or citric acid. A detailed discussion of the many parameters influencing the silver staining reaction can be found in Caetano-Anollés and Gresshoff (1994a). Return
Trouble-shooting
A number of artifacts can occur during electrophoretic separation and silver staining of amplification products. Figure 3 shows examples of such artifacts. In this section I will describe the most common problems associated with different steps of nucleic acid separation and staining and provide trouble-shooting hints.
Polyester film alignment and application
The application of polyester backing film can introduce artifacts during both DNA separation and silver staining. Incorrect film alignment on the bottom of the gel rigs is responsible of the majority of acrylamide leaks during gel pouring. Edge sealing can be accomplished by simply leveling the glass plates, film and spacers along the bottom edge during gel rig assembly. When doing this do not tighten screws forcefully and make sure that the rig components are correctly assembled. If problems persist, Bassam and Bentley (1995) have suggested the use of malleable plasticine adhesive (Blu-Tack, Bostick America, Middleton, MA) as a sealant. Simply press the adhesive along the bottom of the gel rig assembly and snap into position in the casting stand at the time of gel pouring. Remember to remove the original rubber gasket of the stand to make space for the plasticine adhesive.
Incorrect application of the backing sheet to the glass plate can also cause the bottom edge of the film to lift away from the glass. This results in acrylamide depositing behind the film causing uneven gel thickness and irregularities in the electric field during electrophoresis (Fig. 3). Avoid this problem by assembling gel rig components under running water and by pressing the backing film firmly onto the large glass plate. This can be done by rubbing a finger on the film surface and then aligning the rig components. Rig assembly under running water also facilitates alignment of film, glass plates and spacers into position, and eliminates dust and contamination. In particular, avoidance of dust particles (during assembly and gel pouring) eliminates a common type of silver staining artifact, the appearance of spots and streaks in the gels (Fig. 3). The small (front) glass plate makes contact with the gel and should therefore be kept particularly clean and free of scratches.
Gel casting
Use relatively fresh acrylamide stock solutions to attain optimal fingerprint quality. Degassing of the solution is not necessary. However, filter the acrylamide solution through 0.2-0.45 µm filters either when preparing the stock or immediately before casting (see Methods). This eliminates dust particles and the possibility of staining artifacts. The protocols described allow for fast polymerization, produce wells with clearly defined contours, and therefore result in sharper fingerprint bands. Delaying polymerization is not recommended.
Gel loading
To avoid production of irregular wells and aberrant fingerprints, rinse the wells with running buffer immediately after removing the Teflon combs and before loading. This will eliminate residual acrylamide and the chances of polymerization in the wells. A suitable buffer should be used for sample loading (see Methods). Some buffer formulations containing sucrose or glycine were found unsuitable and should be avoided (G. Caetano-Anollés and B.J. Bassam, unpublished). In general, the samples should be loaded swiftly but with steady hand to permit their uniform placement on the bottom of the wells. Avoid introducing contaminants in the wells; they can produce streaking after electrophoresis and staining (Fig. 3). Nucleic acid overloading can also cause streaking and fuzzy bands (Fig. 3). Dilute samples appropriately (usually 1:3-1:10) to avoid these artifacts.
Electrophoresis
Voltages as high as 300 V can be used during vertical PAGE. Depending on running buffer, gel composition and gel thickness, higher voltages can produce band distortion. The use of ultrathin gels in open-faced horizontal PAGE diminish heat generation and permit the use of higher electrical loads. Similarly, the use of long gels in the system increase the tolerance for higher differences in potential (as electrical loads vary with potential drop per cm).
Gel handling and staining artifacts
Following electrophoresis, gel rigs are disassembled, one corner of the backing film lifted, and the backed gel peeled away from the rear glass plate. The backed gels are placed in staining trays (gel facing up), and are fixed and stained with silver. Handle gels with care; they should not touch any surface until image development. During staining, the gel remains attached to the tray by surface tension facilitating removal of spent staining solutions by tipping out the liquid.
To avoid staining artifacts, it is important to wear gloves during gel rig assembly and silver staining. The use of some detergents has been shown to leave residues that increase background staining (Krutchinina and Gresshoff 1995). Clean glass plates with only distilled water, and if necessary use ethanol or an hydrochloric acid wash. Return
RECOVERING AMPLIFIED DNA
Introduction
Individual nucleic acid fragments can be recovered from agarose or polyacrylamide gels by elution from the electrophoretic matrix (Smith 1980). These methods usually require a further purification step by phenol and chloroform extraction, sometimes followed by concentration before subcloning. Fragment isolation is particularly complicated in those cases where a complex assortment of nucleic acids has been resolved. This is the case in a number of applications where PAGE was coupled to silver staining, including DNA sequencing (Doktycz 1993, Storts et al. 1993), single strand conformation polymorphism (SSCP) analysis (Ainsworth et al. 1991, 1993, Dockhorn-Dworniczak et al. 1991, Mohabeer et al. 1991, Maekawa et al. 1993, Sugano et al. 1993), DNA profiling (Allen et al. 1989, Budowle et al. 1991), DAF (Caetano-Anollés et al. 1991), and differential display (DD) of messenger RNA (Lohmann et al. 1995). The majority of these applications are DNA amplification-based and produce a collection of amplification products generally representing one or more discrete portions of a genome. The close proximity of bands in complex DNA profiles, such as those obtained in DAF or DD analysis, makes the isolation of DNA fragments physically demanding. We have developed a simple procedure to recover DNA amplification products from silver stained polyacrylamide gels (Weaver et al. 1994). The method uses one or more rounds of amplification of DNA diffusing passively from gel segments during thermal cycling. Isolated DNA fragments can be used directly without further purification or subcloning. Alternatively, they can be cloned and used in the analysis of DNA polymorphisms and DD expressed sequences or as probes for Southern hybridization, or sequenced and converted into landmarks for genome mapping applications, the so-called sequence characterized amplified regions (SCAR) (Paran and Michelmore 1993). In this chapter I will describe a protocol for the recovery of DNA from polyacrylamide gels and discuss its application to genome analysis. Return
Materials
1. Template nucleic acids (DNA amplification products, including complex mixtures from DAF, DD or sequencing analysis) or silver stained gels.
2. Reaction buffer (10x): 100 mM Tris-HCl, 100 mM KCl (pH8.3).
3. Deoxynucleoside triphosphate 2mM stock solution (dNTPs).
4. Magnesium: 25 or 100 mM magnesium chloride stock solution.
5. Thermostable DNA polymerase enzyme. When using non-stringent amplification conditions, use truncated derivates such as AmpliTaq Stoffel DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT).
6. Oligonucleotide primer(s): 3 or 30 µM stocks.
7. Equipment: thermocycler, bench-top centrifuge, heavy mineral oil, and scalpel. Return
Methods
Isolation of DNA amplification fragments involves their initial separation by PAGE and silver staining (see section 2). Selected DNA fragments embedded in polyacrylamide are isolated directly from the silver stained gels by one or more cycles of isolation and amplification.
1. Carefully excise a small piece of gel containing the desired DNA fragment from the gels with a flamed scalpel or dissection probe.
2. When using a preserved gel, clean the surface of the gel with 95% ethanol, use the scalpel to sharply delimit the segment of interest, and then rehydrate the excised band with a drop of sterile water.
3. Place the gel segment in 10-100 µl of standard amplification mixture containing 0.3-3 µM of primer(s), 0.3 units/µl of AmpliTaq Stoffel fragment, 1.5 mM magnesium chloride, 200 µM of each dNTP, and buffer.
4. Centrifuge, and if necessary, cover the amplification mix with 1-2 drops of heavy mineral oil.
5. Heat for a minimum of 5 min at 95C if the fragment to isolate is larger than 500 bp.
6. Generally, amplify in 35 cycles of 30 s at 96C, 30 s at 50C and 30 s at 72C in an oven thermocycler (Bios, New Haven, CT). Several amplification regimes can be used depending on primers and stringency required.
7. Resolve amplification products using PAGE and silver staining.
8. Alternatively, bands can be eluted from the gel segments at 95C for 20-30 min and the recovered eluate used as template in step 3. Return
Comments
We have shown that silver stained polyester-backed polyacrylamide gels act as safe repositories of nucleic acids (Weaver et al. 1994). The gels can be preserved dried for many years without suffering distortion or image loss, allowing the retrospective analysis of the embedded nucleic acid molecules. The desired silver stained bands are recovered from the polyacrylamide matrix by one or more rounds of band excision, amplification, and electrophoretic analysis. Gel segments are used directly as template in each amplification reaction.
We have isolated many bands representing interesting monomorphic or polymorphic DAF products from bacterial, fungal and plant fingerprints (eg., Caetano-Anollés et al. 1992, 1993, Weaver et al. 1994, 1995). Silver stained products were recovered from profiles separated using vertical and horizontal PAGE, discontinuous polyacrylamide isotachophoresis, and the PhastSystem, under both denaturing and nondenaturing conditions. The procedure was efficient and selective enough to remove the unwanted contaminants, even if the isolated bands were relatively minor amplification products and were very close in molecular weight to abundant products in the profile.
Number of recovery cycles
The number of amplification recovery cycles depends mostly on the complexity of the DNA pattern from where a particular fragment is isolated. Recovery occurs in a single cycle when bands are isolated from PCR products, even if resulting from multiplex PCR reactions. In contrast, at least two cycles are necessary to recover bands from complex profiles such as those generated in DAF or DD analysis. During recovery and in few instances, minor contaminating product persist at very low levels even after several isolation cycles. These contaminants usually represent only a small fraction (about 5%) of recovered DNA and are highlighted by the sensitivity of nucleic acid silver staining.
Factors affecting recovery
Several factors influence the recovery of nucleic acids, especially polyacrylamide concentration, DNA polymerase and annealing temperature used during amplification, and the length of isolated fragments. Higher polyacrylamide concentrations appear to decrease the number of recovery cycles, most probably by decreasing product carryover during electrophoresis (Weaver et al. 1994). For example, the isolation of a soybean DAF marker required 5 cycles when using 4.5% polyacrylamide gels, but only 3 cycles when using 6% gels. In SSCP analysis, band isolation from 20% polyacrylamide gels demanded thorough washing of the silver stained gel prior to band excision and the use of high number of temperature cycles during recovery (Calvert et al. 1995).
Band recovery also appears strongly conditioned by the DNA polymerase used during amplification (Weaver et al. 1994). When using some variants of Thermus aquaticus DNA polymerase, such as the truncated Klentaq LA (AB Peptides, St. Louis, MO), succesive rounds of band recovery and amplification were unable to eliminate contaminating products. One of possible explanations is the very efficient amplification of contaminating template DNA, even if present at very low levels in the excised gel segments.
High annealing temperature and therefore high stringency amplification helps eliminate many contaminants during band recovery. For example, the number of contaminants still remaining after a first cycle of isolation can be halved if annealing temperature was raised from 30C to 50C. In higher stringency is required, a "hot-start" can enhance relative enrichment and fragment recovery.
Finally, the length of the amplification fragments to be isolated is an important consideration. Generally, small fragments are readily isolated, the shorter primers requiring a lower number of recovery cycles. In contrast, DNA fragments of more than 600 bp can only be isolated if gel segments are incubated at 95C for at least 20 min prior to amplification (Sanguinetti et al. 1994). Obviously, long fragments should be given enough time to diffuse out of the gel segments. Furthermore, it is recommended that only a small gel segment be excised (to minimize inhibitory effects of silver or silver-staining components; Calvert et al. 1995) and that each round of isolation be given at least 25-30 temperature cycles to maximize amplification.
Applications
Individual amplification products isolated from silver stained polyacrylamide gels can become probes for Southern hybridization (Caetano-Anollés et al. 1993, Weaver et al. 1994,1995). Isolated DAF products can be used directly in hybridization studies without further subcloning (Weaver et al. 1994), either to confirm their monomorphic or polymorphic nature when hybridized to DAF profiles, or to determine if the originating DAF products represent single or multi-locus sites in the genome when hybridized to genomic DNA. Nevertheless, isolated fragments can be cloned without further purification using either "blunt-ended" or "overhanging-ended" cloning strategies (see Frohman 1994), especially when purity of the selected fragment is mandated or when isolated products are to be sequenced. Overhanging-ended cloning is considered the best alternative, though in some cases it requires the use of primers encoding restriction endonuclease sites. Ultimately, recombinant plasmids are amplified by the PCR, and cloned fragments radioactively labeled for hybridization or sequenced with forward and reverse primers complementary to sites in the cloning vector (Sambrook et al. 1989).
Recovery of amplification products from silver stained gels may be especially important whenever retrospective examination of genetic evidence is entered in a court of law, such as in forensics, plant variety rights enforcement, and parentage testing. Isolated bands can be used as hybridization probes in many applications, including those in marker-assisted breeding, genetic mapping, general fingerprinting, molecular ecology and evolution, and gene expression studies. Alternatively, isolated bands representing interesting polymorphic loci can be converted into amplification-derived sequence-tagged sites. For example, isolated DNA fragments originating from the amplification of DNA with arbitrary primers can be converted into SCARs (Paran and Michelmore 1993). SCARs are PCR-based markers that are especially useful for genetic mapping and positional cloning. SCAR amplification is usually driven by PCR primers complementary to the ends of an isolated amplification fragment. SCARs retain the original dominant segregation behavior of the originating marker or become co-dominant. Dominant SCARs can be converted to co-dominant SCARs by digestion with restriction endonucleases, or fingerprinting with mini-hairpin or microsatellite primers. However, useful SCARs must be polymorphic in a mapping population and defined as a single locus by segregation analysis. Only then can they be used as landmarks for physical mapping, as anchoring points between physical and genetic maps, and in comparative mapping of related species. SCARs can be used to screen genomic libraries, construct contigs, and identify overlapping clones in chromosome walking. Return
