Introduction
Principles
Materials
Methods
DAF analysis with mini-hairpin primers
Arbitrary signatures from amplification profiles (ASAP)
Template endonuclease cleavage MAAP (tecMAAP)
Post-amplification procedures
Comments
Mini-hairpin primers
ASAPs: fingerprints of fingerprints
tecMAAP: predigestion with restriction endonucleases
In many nucleic acid scanning applications there is a need to tailor the performance of the amplification reaction. For example, in molecular ecology and evolution there is sometimes a requirement to increase or decrease the ability to distinguish a group of organisms. Generally, this is done by using more than one fingerprinting technique capable of resolving genomes, for example, at the species or subspecies level. The versatility of nucleic acid scanning has overcome some of these limitations. The concept of "fingerprint tailoring" was introduced some time ago to depict those strategies which modify fingerprint pattern. Tailoring can target the number and range of amplification products, the level of polymorphic DNA detected, template complexity, and even the nature of amplified sites. For example, detection of DNA polymorphisms and profile complexity (multiplex ratio) can be enhanced by digestion of template DNA with several type II restriction endonucleases that bind to 4 bp recognition sequences (tecMAAP) (Caetano-Anollés et al. 1993), when using unique mini-hairpin primer chemistry (mhpDAF) (Caetano-Anollés and Gresshoff 1994), or when producing arbitrary signatures from amplification products (ASAPs) (Caetano-Anollés and Gresshoff 1996). This section describes some of these fingerprinting alternatives. Return
Principles
Fingerprint tailoring is based on improving: a) analysis of amplification products, b) primer design, and c) amplification strategy (Caetano-Anollés 1996). The way how amplification products are studied impacts on the outcome of fingerprint pattern. In DAF, some amplification products can only be detected when more sensitive separation techniques are used. For example, capillary electrophoresis promises to increase throughput but also detection of polymorphic DNA (Caetano-Anollés et al. 1995). While the capabilities of analyzing fingerprints are confined to the actual originating amplification process, the tailoring of primer design and of amplification strategy is much more versatile. Primers can be tailored to produced DNA fingerprints of desired complexity by designing their sequence and ultimately the specificity of annealing to their targeted sites. The selection of primer sequence can be arbitrary and based on preliminary experience (ie. choosing those primers that work best), or can be biased to introduce secondary structure within certain primer domains (e.g., mhpDAF), recognize particular sequence motifs within the genome that represent dispersed sequences, structural chromosomal domains or even consensus sequences complementary to gene families, or include degeneracy at particular locations within primer sequence. Tailoring can also be accomplished by improving the amplification strategy, such as in the case of template endonuclease cleaved MAAP (tecMAAP) (Caetano-Anollés et al. 1993; see section 4.5.3) and AFLP analysis (Vos et al. 1995).
While tailoring changes the complexity of fingerprint pattern it can also increase the percentage of amplification products that are polymorphic within a particular group of organisms or templates under study. Two major mechanisms are responsible for such an effect: a) an increase of the number of sites being probed in the template, for example during primer annealing, endonuclease restriction or post-amplification manipulations, and b) a change in the kinetics of the reaction due to novel primer-template interactions or changes in the stringency of amplification. Return
Materials
1. Reaction buffers (10x stocks): Stoffel (STF) buffer, 100 mM Tris-HCl, 100 mM KCl, pH 8.3; TTNK10 buffer, 200 mM Tris-HCl, 1% Triton X-100, 40 mM ammonium sulfate, 100 mM KCl, pH 8.6.
2. Deoxynucleoside triphosphate stock: 2 mM of each dNTP.
3. Magnesium solution: 25 mM magnesium chloride (for use with STF buffer) or 100 mM magnesium sulfate (with TTNK10 buffer).
4. Oligonucleotide primers: 30 µM or 300 µM stock solutions.
5. AmpliTaq Stoffel fragment DNA polymerase (Perkin-Elmer, Norwalk, CT).
6. Template: 1-50 mg/µl stock solutions. Return
Methods
The following are protocols of MAAP techniques capable of fingerprinting a wide variety of templates, including plasmids, cloned DNA, and PCR products, and increasing detection of polymorphic DNA. General precautions and considerations described for DAF analysis should also be applied to these techniques (see DAF).
DAF analysis with mini-hairpin primers
Primers containing hairpin-turn structures at their 5' termini can generate reliable fingerprints from almost any template nucleic acid. However, the design of the amplification reaction varies with template complexity.
1. Assemble an amplification cocktail in 10-20 µl total volume containing the following components: 0.1-5 ng/µl template DNA, 3-30 µM of primer(s), 0.3 units/µl of thermostable DNA polymerase, 200 µM of each deoxynucleoside triphosphate, 4 mM magnesium sulfate, and TTNK10 buffer.
2. Cover the amplification cocktail with 1-2 drops of heavy mineral oil, when required.
3. Amplify in 35 cycles of 30 s at 96C, 30 s at 30C and 30 s at 72C in an oven thermocycler (Bios, New Haven, CT).
4. Retrieve the aqueous phase from reaction tubes.
5. Dilute samples 5-10 fold prior to analysis.
Arbitrary signatures from amplification profiles (ASAP)
ASAPs are "fingerprints of fingerprints" that result from the reamplification of DAF products with one or more arbitrary primer harboring a sequence that differs significantly from that of the primer used to generate the original DAF reaction (Caetano-Anollés and Gresshoff 1996). Generally, primers used in the second round of amplification are mini-hairpin oligonucleotides, though unstructured DAF primers or primers complementary to interspersed repetitive sequences can be used. The following protocol is designed for used with mini-hairpin primers.
1. Dilute DAF reactions, usually containing 100-200 ng/µl of double stranded DNA, to about 1 ng/µl template (10x) stock solution. Typically, this corresponds to a 1:100 dilution of a standard amplification reaction.
2. Assemble the amplification cocktail in 10-20 µl total volume containing the following components: diluted amplification products (0.1 ng.µl final concentration), 9 µM of primer(s), 0.3 units/µl of thermostable DNA polymerase, 200 µM of each deoxynucleoside triphosphate, 4 mM magnesium sulfate, TTNK10 buffer, and diluted amplification products.
3. Cover the amplification mixture with 1-2 drops of heavy mineral oil, when required.
4. Amplify in 35 cycles of 30 s at 96C, 30 s at 30C and 30 s at 72°C in an oven thermocycler.
4. Retrieve the aqueous phase from reaction tubes.
5. Dilute samples 5-10 fold prior to analysis.
Template endonuclease cleavage MAAP (tecMAAP)
tecMAAP is based on a two-step reaction where the template nucleic acid is first subjected to enzymatic digestion with type II restriction endonucleases and then amplified with one or more arbitrary oligonucleotide primer.
1. Digest template DNA with one or more restriction endonucleases. Add 2 Units of enzyme (usually 2 µl) and 2 µl of appropriate restriction enzyme buffer (Table 1) to 2 µg of DNA in a total 10 µl reaction volume. Incubate at the recommended temperature for 1 h to overnight. Both blunt-end or staggered-cutter enzymes can be used. However, enzymes that recognize 4 bp motifs are preferred.
2. Confirm complete digestion by electrophoresis in agarose or polyacrylamide gels.
3. Assemble an amplification cocktail in 10-20 µl total volume containing the following components: 0.1-5 ng/µl digested template, 3-30 µM of primer(s), 0.3 units/µl of thermostable DNA polymerase, 200 µM of each deoxynucleoside triphosphate, 1.5 mM magnesium chloride, and STF buffer. Use MgSO4 and TTNK10 buffer when amplifying with mini-hairpin primers.
4. Cover the amplification cocktail with 1-2 drops of heavy mineral oil, when required.
5. Amplify in 35 cycles of 30 s at 96C, 30 s at 30C and 30 s at 72C in an oven thermocycler (Bios, New Haven, CT).
6. Retrieve the aqueous phase from reaction tubes.
7. Dilute samples 5-10 fold prior to analysis.
Post-amplification procedures
Amplification products can be separated by polyacrylamide gel electrophoresis (PAGE) in vertical or open-faced slab gels, or using semi-automated miniaturized electrophoretic devices (see section 2). Gels are backed on polyester film, silver stained using the procedure of Bassam et al. (1991), and preserved by drying at room temperature. Return
Comments
Mini-hairpin primers
A straightforward approach to tailor fingerprints is the improvement of primer design. To do so, one can take advantage of how primer length, primer sequence and secondary structure influence the amplification reaction. For example, very short primers (5-6 nt) produce relatively simple DAF profiles that resemble those obtained in RAPD analysis. In this case, amplification with short primers is hampered by: a) the existence of palindromic termini in amplification products capable of forming hairpin loops, as the short primer has great difficulty in displacing these hairpin-structures (Caetano-Anollés et al. 1992), and b) an inherent decrease of primer annealing efficiency with decreasing primer length. However, this effect can be offset with the introduction of an extraordinarily stable and compact hairpin-turn structure at the 5' end of the primer that in some way interferes with the formation of hairpin loops in the resulting amplification products (Caetano-Anollés and Gresshoff 1996). These mini-hairpin primers can therefore harbor a very small "core" arbitrary sequence at their 3' termini, and still produce complex and reliable "sequence signatures", even from small template molecules such as plasmids, cloned DNA, and PCR amplified fragments.
Mini-hairpins are short DNA segments can form an extraordinarily stable structure consisting of a loop of 3-4 nt and a 2 nt stem (Hirao et al. 1988, 1989, 1992; see Figure). Mini-hairpins have high melting temperatures, unusually rapid mobilities during electrophoresis in polyacrylamide gels, and cause band compression during Maxam-Gilbert DNA sequencing. Their extraordinary stability depends on the existence of a hairpin-turn structure determined by the helical motif of the stem region, the loop-closing base pair, and a balance between bendability and stacking stability of a B form structure in the loop (Hirao et al. 1994). They have been observed in natural DNA, such as in the replication origin of phage G4 or in rRNA genes (Hirao et al. 1989, 1990, Antao and Tinoco 1991a,b). Mini-hairpin primers are designed by attaching a 7-8 nt hairpin-turn structure (the mini-hairpin) to the 5' end of a "core" oligonucleotide of arbitrary sequence. These oligonucleotides retain the characteristics of the mini-hairpin, such as high stability, high melting temperatures and rapid electrophoretic mobilities.
Amplification with mini-hairpin primers has been optimized (Caetano-Anollés and Gresshoff 1994) using a buffer containing Triton X-100 and ammonium sulfate (Caetano-Anollés et al. 1994). Amplification requires 2-12 mM magnesium sulfate concentrations, with an optimal concentration range of 3-6 mM. Primers with long core regions are less tolerant of high magnesium levels. A minimum primer concentration of 1.5 mM is generally sufficient for reproducible amplification. However, higher primer concentrations (up to 30 µM) increase the efficiency of amplification but do not alter profile composition. In the study of PCR fragments, plasmids or viral DNA, reproducible patterns can only be obtained using high primer concentration (30 µM). Template DNA concentrations should be comparable to those used in standard DAF. Optimal template concentration usually ranges 0.1-5 ng/µl.
Mini-hairpin primers have the property of increasing detection of polymorphic DNA when fingerprinting genomic DNA from a variety of organisms, including plant species such as centipedegrass (Caetano-Anollés and Gresshoff 1994, Weaver et al. 1995), bermudagrass (Caetano-Anollés et al. 1995), and soybean (Caetano-Anollés and Gresshoff 1996). For example, the ability of mini-hairpin primers to differentiate bermudagrass cultivar 'Tifway' from its gamma-irradiation mutant, 'Tifway II', was increased almost 5-fold when compared to the use of standard primers (Caetano-Anollés et al. 1995). The enhanced resolving power of mini-hairpin primers may result from an increase in the size of the genome being probed during annealing. As discussed above, annealing of mini-hairpin primers appears influenced by secondary structure of DNA and interactions between amplicon termini. Generally, the scanning of extended annealing sites increases the chances of finding sequence variation. Mini-hairpin primers may accomplish this by targeting a high number of anealing sites with their short arbitrary cores and then selecting amplicons with extended annealing sites at their termini during the scanning of first-round amplification products.
ASAPs: fingerprints of fingerprints
As originally described, ASAPs are fingerprints generated from fingerprints by re-amplification of DAF profiles with mini-hairpin or standard arbitrary primers (Caetano-Anollés and Gresshoff 1996). ASAPs are also fingerprints obtained by re-amplification of any amplification product, ranging from those generated with arbitrary primers to those produced in the PCR.
ASAP analysis is a dual-step amplification strategy that provides additional scanning of primary sequence within preselected amplicons. The procedure requires the use of one or more primers in each amplification step, allowing the combinatorial use of oligomers in fingerprinting. Provided the sequence of the primers differ substantially from each other, distinct fingerprints can be generated in each particular combination. For example a set of 10 oligomers can be used in 100 different pairwise combinations, 90 of which should produce unique fingerprints. The power of the approach is further unveiled when multiplexing DAF reactions. If two primers are used in one of the amplification steps, or in both, 104 or 1016 different reactions are possible, respectively. However, priming during the second amplification occurs within the DAF fragments amplified in the first amplification. Therefore, the number of targeted locations in a genome depends on the number of original DAF reactions.
The arbitrary selection of primers in ASAP analysis can be biased to include recognition of particular sequence motifs or interspersed repetitive sequences. For example, primers complementary to simple sequence repeats (SSR) present in microsatellite loci can be used to generate very simple ASAPs by re-amplification of MAAP fingerprints (Caetano-Anollés and Gresshoff 1996). The advantage of targeting these loci is that they represent highly polymorphic regions, and in some cases can constitute co-dominant markers with many allelic forms. The high informativeness of SSR loci has made them the target of a number of MAAP derived techniques, where primers complementary to the sequence repeats are used directly or with arbitrary 5' or 3' terminal anchors in an amplification reaction of genomic DNA (Meyer et al. 1993, Perring et al. 1993, Zietkiewicz et al. 1994, Wu et al. 1994). Unfortunately, all these techniques amplify both the SSR motif and unrelated arbitrary sequences, and generate fingerprints with relatively high multiplex ratios where co-dominance may be difficult to interpret (Weising et al. 1995). The simple profiles of SSR-ASAP analysis mitigates the problem by generating very simple profiles containing in 50% of the cases only 1-2 prevalent amplification products. These products represent SSR loci and in many cases are clearly co-dominant. In order to target consistently about 10-15 kb of DAF amplified sequence which contains only few SSR annealing sites, the SSR primers have to be anchored at their 5' termini with ambiguous (ie. degenerate) nucleotides. Furthermore, the amplification reaction must occur under stringent conditions to avoid mismatch priming.
ASAP analysis relies on finding appropriate "reproducibility windows" (see section 1) for the many parameters in the amplification reaction, especially primer and template concentration. Primer concentration was found particularly important for reproducibility (Caetano-Anollés and Gresshoff 1996). Mini-hairpin decamers require 6-9 µM concentrations, while standard octamers require at least 9 µM primer levels. ASAP primers with sequences partially complementary to the termini of DAF products tolerate lower primer concentrations (about 3 µM). The reproducibility window for template concentration ranges 0.001-1 ng/µl of DAF products, even if ASAP primers are mini-hairpins partially complementary to the DAF primers used in the first amplification reaction. However, a decrease in primer concentration during ASAP amplification causes the window to narrow considerably. It is therefore important to consider the existence of primer-template interactions in new ASAP fingerprinting applications.
tecMAAP: pre-digestion with restriction endonucleases
tecMAAP couples MAAP to endonuclease cleavage of the template prior to amplification (Caetano-Anollés et al. 1993). Generally, one or more restriction endonucleases (preferably three 4 bp cutters) are used to restrict the DNA prior to amplification. This DNA then serves as a modified template for amplification with arbitrary primers. The strategy is straightforward but contingent on the assurance that template digestion has been complete.
tecMAAP has been shown to enhance considerably (up to 100-fold) the detection of polymorphic DNA, alowing the identification of closely related cultivars, plant accesions, and even near-sogenic lines. During MAAP, many sites are targeted but only few are preferentially amplified in a dynamic reaction where reaction kinetics and primer-template interaction determine the outcome of a particular fingerprint. In tecMAAP, digestion eliminates many of possible amplicons and ultimately reduces the effective length of template DNA. Restriction also increases the nucleotide sequence being probed. Sequence variation within restriction sites will either directly eliminate some of preferentially amplified products, or will indirectly change the overall kinetics creating new products or eliminating ones that were previously amplified. Computer simulation has been applied to this problem to show that upon restriction, arbitrary octamer primers are forced to mismatch with the template in the last one or two 5' terminal nucleotides (Caetano-Anollés 1994). Ultimately, differential cleavage of template molecules enhances the detection of polymorphic DNA without changing appreciably the multiplex ratio. Return
