Amplifying DNA with Arbitrary Ollgonucleotide Primers

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Gustavo Caetano-Anoll~s

Plant Molecular Genetics, Institute of Agriculture and Center for Legume Research, The University of Tennessee, Knoxville, Tennessee 37901-1071

Genetic molecular markers are DNA terminate processes that target multiple eny populations can be measured by the segments that behave as landmarks for sites of defined sequence in both DNA variation in length or sequence of DNA genome analysis. These segments usu- strands. In contrast, amplification with segments; however, the identification of ally represent variant or polymorphic degenerate primers in random inde- these molecular markers requires prior sites that can be identified using general terminate amplification processes, knowledge of DNA sequence, cloned and strategies such as molecular hybridiza- like random primed amplification characterized probes, and considerable tion or enzymatic amplification of DNA. (RPA), (2~ primer-extension preamplifi- experimental manipulation. These limi- For years, DNA-based diagnostic mark- cation (PEP),(21) and random PCR tations can be circumvented with multi- ers have been used in general organismal (rPCR), (22) take advantage of stochastic ple arbitrary amplicon profiling identification and in the construction of annealing events that randomly amplify (MAAP)(30) techniques like random genetic linkage maps. (1) The widely used nucleic acid stretches or even whole ge- amplified polymorphic DNA (RAPD) restriction fragment length polymor- nomes by targeting sites of a noncognate analysis, (24) arbitrarily primed PCR phisms (RFLPs), for example, are molec- nature. These random DNA amplifica- (AP-PCR), (zs) and DNA amplification fin- ular markers identified by endonuclease tion procedures are generally used for ra- gerprinting (DAF).(26) These indepen- restriction and blot hybridization of dioactive or fluorescent labeling of nu- dently developed strategies use one or DNA. (z) DNA amplification using cleic acids, to increase the amount of more arbitrary oligonucleotide primers PCR (3'4) has also been used extensively DNA in the construction of representa- to target specific but unknown sites in in many applications to study poly- tive cDNA libraries, or for PCR typing of the genome, many of which are polymorphic loci, like hypervariable minisat- single haploid cells. Other kinds of am- morphic. Detected amplification frag- ellites (s) or microsatellites harboring plification processes, although still de- ment length polymorphisms (AFLPs) can simple sequence repeats, (6-8) and to gen- terminate, do not require prior knowl- be used easily as markers for genetic typ- erate sequence-tagged sites (STSs)(9) for edge of template sequence and can ing and mapping applications. (31'32) genetic and physical mapping. target single or multiple sites in a ge- MAAP techniques target multiple and Several strategies involving DNA rep- nome or template molecule. These strat- arbitrary amplicons but differ in primer lication, DNA ligation, or RNA transcrip- egies can use either one arbitrary primer length, amplification stringency, and tion have been used for in vitro amplifi- in conjunction with a specific primer (23) procedure used to resolve DNA patterns. cation of nucleic acids. (1~ However, or one or more arbitrary primers (24-26) to For example, AP-PCR(2s) uses primers of PCR remains the most widely accepted study single or multiple amplicons, re- lengths comparable with those of PCR amplification tool. Primer-directed am- spectively, and can even be extended to primers, RAPD(24) analysis uses shorter plification of DNA, at first used in PCR to the analysis of RNA populations. (27-28) primers, typically 10 nucleotides in amplify cognate regions present at very In particular, strategies like gene-walk- length, and DAF(26) uses primers as short low levels in the genome, has extended ing PCR (23) and differential cDNA as 5 nucleotides, but typically 7 or 8 nu- DNA analysis to regions adjacent to se- PCR (27) study specific template regions cleotides in length. These and other dif- quenced DNA segments, to unknown arbitrary in length but juxtaposed to a ferences result in DNA profiles that are DNA, and even to the study of RNA-ex- known DNA segment defining a specific dearly distinct. Whereas RAPD resolves pressed sequences. (~s-~7) Amplification primer. Like anchored PCR,(29) these few amplification products using agar- strategies can be grouped according to hemispecific reactions allow analysis of ose gel electrophoresis and ethidium the mechanism of the amplification pro- unknown genomic regions correspond- bromide staining, DAF produces charac- cess (Table 1). Amplification with spe- ing to mRNA sequences, or adjacent to teristic and complex information-rich cific primers in the PCR, for example, is STSs or sequenced stretches of DNA. DNA profiles using polyacrylamide gel a determinate process that requires prior electrophoresis and a silver stain that de- knowledge of the template sequence and tects DNA at the picogram level. (33) Fin- ANALYSIS OF MULTIPLE AMPLICONS targets usually one defined amplifica- gerprints are determined by primer an- OF ARBITRARY NATURE tion site. Similarly, amplification of in- nealing to partially or perfectly terspersed repetitive sequences (IRS), The degree of relatedness between indi- complementary sites on each DNA like Alu-PCR (18) or REP-PCR, (19) are de- vidual organisms or inheritance in prog- strand. Therefore, primer design, ampli-

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TABLE 1 DNA Polymerase-mediated Nucleic Acid Amplification

Amplification Primer process Amplicon number annealingb target (site/strand) Strategy a Determinate single two or more defined/S specific/one each PCR single two defined and arbitrary/P specific/one each gene-walking PCR multiple one or more defined/S specific IRS/both AIu-PCR,REP-PCR, ERIC-PCR multiple one or more arbitrary/S,P arbitrary/both DAF, RAPD, AP-PCR Indeterminate multiple degenerate stochastic/P random/both RPA, PEP, rPCR aAmplification strategies that require additional processes, like vectorette ligation, whole genome PCR, or key PCR, are not listed. b(S) Stringent; (P) permissive. Amplification stringency is defined by reaction conditions (like annealing temperature or primer concentration) that favor specificity during the different cycles of amplification. fication reaction conditions, and ade- diphosphate from deoxynucleoside the DNA for possible annealing sites and quate separation and detection of ampli- triphosphates. to anneal to many of them. Following fied products temper the reproducibility, Figure 1 depicts the reaction mecha- this "amplicon screening" phase, the complexity, and polymorphic content of nism proposed by Caetano-AnollEs et thermostable DNA polymerase then an- DNA profiles. (34) For example, RAPD al. (3s~ to explain how a single primer am- chors and initiates the extension of the analysis using decamer primers pro- plifies a target genome. The first ampli- annealed primers, producing a defined duced fingerprints that were on average fication cycle begins by denaturing the population of "first-round" amplifica- 3-10 times less complex than those gen- template DNA molecules. Decreasing tion products. The discrimination be- erated using DAF with decamer and oc- temperature allows the primer to scan tween cognate and noncognate primer- tamer primers. (34) Superficial versus exhaustive examination of amplification products may be partially responsible for differences in profile complexity. However, primer, magnesium, and deoxynucleoside tri- phosphate concentration, as well as temperature cycling parameters and other amplification reaction components, can alter reaction stringency and dramati- cally influence both the number and the nature of amplification products.

REACTION MECHANISM

DNA polymerase-mediated nucleic acid amplification is a complex and dynamic series of chemical reactions where primers single out DNA segments and determine the efficiency of their subsequent amplification. The balance between template, amplification products, enzyme, primers, and deoxynucleoside triphos- phate concentration changes with each temperature cycle as amplification products are synthesized and different molecular species interact with each other. Al- though DNA amplification is most likely a reaction process departing from equi- FIGURE 1 Model of interactions between molecular species formed during DNA amplification librium, it may be argued that in every with a single arbitrary oligonucleotide primer. During a first template "screening" phase ($1, $2) and depending on amplification parameters, cognate and nocognate sites compete and are se- cycle of amplification there are two lected for amplification ($3). The synthesized first-round amplification products ($4) are initially reaction components, a standard single stranded and have palindromic termini. In subsequent rounds of amplification, the prod- Michaelis-Menten kinetic process dis- ucts can establish template-template (A1, A2) and primer-template (A3, A4) interactions. Some criminating target cognate DNA from interactions may occur late during amplification (A6, A7). The different species produced tend to noncognate competitor sites, and a vir- establish an equilibrium. Whereas enzyme anchoring and primer extension (A5) transform the tually irreversible process that is thermo- relatively rare primer-template duplexes into accumulating amplification products. In an ideal dynamically driven by the cleavage of MAAP amplification, S1 >> $2, and A3 >> A1 >> A6.

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template interactions during the first Inefficient extension of primer-template length, to study primer-template inter- temperature cycles determines the num- mismatches appears to be caused by actions established during DAF.(3s) The ber and the nature of amplicons selected. failed DNA elongation rather than by a short oligonucleotides used in these ex- A change in the concentration of template differential enzyme binding affinity of periments approached their functional DNA or primer will obviously affect ampl- matched versus mismatched termi- limits as primers for amplification. Our icon screening, although other compo- ni. (36-39) In structural crystallographic study defined several domains in the nents may also influence the reaction. studies of the Klenow fragment, 11 nu- primer-template duplex (Fig. 2). The When amplicons are defined by two cleotides of a primer-template duplex first 8 nucleotides from the 3' terminus primers, first-round amplification prod- were positioned in a groove that lies at of the primer encompassed a region ucts usually become preferred templates right angles to the cleft containing the largely responsible for directing the am- and are subsequently amplified with polymerase active site and adjacent to plification process. Single base changes high efficiency. However, single primer the 3' --, 5' exonuclease domain. (4~ in sequence within this domain altered amplification products have palindro- These and other studies (41'42) suggest the spectrum of amplification products mic termini and, when present in low that duplex DNA containing the primer significantly, especially toward the 3' concentration, can produce hairpin loop 3' terminus is distorted and is close to terminus. Results show the importance structures. Because the primer must dis- the exonuclease site, forcing the primer of the 3'-terminal region of the primer in place these hairpin loop complexes long terminus to shuttle between active sites, the DAF reaction and confirm observa- enough for the enzyme to anchor and whereas the single-stranded template en- tions for PCR.(49) stabilize the duplex by strand extension, ters from the distal end of the cleft. Mis- Whereas successful amplification re- and because the extent of hairpin loop matches in some of the 19-20 nucle- quires a primer of at least 5 nucleotides interference will be variable for each otides of the primer-template duplex in length (26) and annealing sites with fragment, only some of the first-round proposed to interact with the enzyme (41) perfect homology to the first 5 or 6 nu- amplification products will be efficiently are expected to alter extension effi- cleotides from the 3' terminus, (34) de- amplified. ciency. These primer-template mis- creasing primer length within the 8-nu- During late amplification cycles, at- matches must be identified if we are to cleotide domain results in an tenuation in the rate of product accumu- understand interactions that drive DNA unpredicted decrease in the number of lation turns exponential amplification amplification. amplification products and in the pro- into a linear process. This "plateau" phe- The number and location of primer- duction of highly variant fingerprint pat- nomenon occurs when the increasing template mismatches (especially if they terns. In theory, primers of increasing mass of preferred amplification products occur at the 3' terminus of the primer), length derived from a common se- limits the activity of the polymerase en- their stability at various annealing and quence should target subsets of sites rec- zyme, reduces the efficiency of template extension temperatures, and the effi- ognized by shorter primers, and a com- denaturation, increases the 5' --~ 3' sin- ciency with which the polymerase can mon pattern should dominate all gle-stranded exonuclease activity of DNA recognize and extend the mismatched fingerprints. As this was not the case, re- polymerase, and decreases primer an- duplexes have been studied extensively suits suggest that only a fraction of tem- nealing by favoring the formation of in the PCR process. (38'43-49) However, plate-annealing sites amplify effi- template duplexes. The high concentra- factors such as the effects of primer ciently. (3s) Furthermore, certain tion of amplification products also fa- length, primer and template conforma- amplicons are amplified preferentially vors reannealing events between the var- tion, and reaction environment still re- by short primers due to competition for ious template molecules. Because DNA quire careful examination. We used reannealing sites between primer and ter- template species are independently am- lated oligonucleotides differing in minal hairpin loop structures of the tem- plified, amplicons that have been ampli- length or sequence, and templates engi- plate. (3s) Primers 8 nucleotides or longer fying at low levels are now selected as neered to have complementary or mis- were better able to complete for anneal- preferred templates. Therefore, primer- matched terminal sequences of varying ing with hairpin loops. template interactions become crucial in determining specificity, not only in the presence of a vast molar excess of an- 5' domain 3' domain nealing sites during amplicon screening ) 4 9 but also late during amplification when underrepresented amplicons are the only molecular species that are being ex- ponentially amplified. 3'1 Io

UNDERSTANDING PRIMER-TEMPLATE INTERACTIONS II Minimal sequence The preferential amplification of an am- .:.:.:.:.:.:.:.:.: ili~i~!~!~!~i~i~i~~ plicon is driven by the interactions of ~=~=~? Mismatch tolerance the primer with its annealing site, fol- FIGURE 2 Primer-template duplex domains established during amplification of template DNA lowed by appropriate recognition of with arbitrary primers. The arrow indicates the direction of primer extension by DNA polymerase those interactions by DNA polymerase. (see text for details).

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Regions beyond the basic 8-nucle- cause long 5'-terminal primer domains tion of bona fide products. However, otide domain alter the amplified spec- may contain regions of partial homol- varying time intervals, annealing, exten- trum only moderately. (3s~ In particular, ogy to the template, favoring some am- sion or denaturation temperature, cycle the domain is demarcated by a 2- to plicons over others. numbers, or even heating and cooling 3-nucleotide region over which changes Other primer characteristics also de- rates can complicate the uniform and in primer length do not alter fingerprint termine primer-template interactions. simple usage of an amplification tech- pattern (Fig. 2). Increasing the primer Using templates with ends engineered to nique. Therefore, other parameters like length does not decrease the number of have perfect homology to the 3' termi- primer, template, and ion concentra- amplification products. This and the fol- nus of the primer, we determined that tions, or the DNA polymerase itself, lowing lines of evidence suggest primer- overhangs in both strands of primer- must be considered. template mismatching at the 5' end of template duplexes (produced as a result of differences in primer length) affected the primer. First, amplification of ge- Reaction Components and the formation of many amplification nomes of high complexity like soybean Environment and human with primers of >8 nucle- products. ~3s~ Similarly, fluorophore la- otides in length and of bacteria and bels and stable minihairpins at the 5' ter- Using defined primer-template combi- fungi with primers of >7 nucleotides in minus of the primer altered fingerprint nations, we followed an iterative process length produce many more products patterns. ~ss~ to optimize the DAF reaction carefully than expected. ~34~ Similarly, primers Within a 50-100% GC content, we for genomes of high (6~ and low com- comparable with those used in PCR pro- have not found any influence of frac- plexity. ~ss~ Similarly, careful optimiza- duced AP-PCR and RAPD fingerprints tional primer GC content on amplification has preceded several studies using from bacterial, fungal, plant, and animal tion. (34) Recently, Fritsch et al. ~s6~ sur- RAPD analysis. (s2-s4'61-63) Fingerprints species, (2s,s~ where no products were veyed 480 decamers and found that total obtained with different eubacterial DNA to be expected on theoretical grounds (34~ GC content had the most predictive polymerases vary considerably and re- (unless the primer recognizes disperse value in estimating the efficiency of quire widely different optima for the var- repetitive sequences). Second, primers of RAPD amplification. Their results con- ious amplification parameters. Gener- 8 and 10 nucleotides in length produced firmed the previously observed direct ally, truncated DNA polymerases like identical fingerprint patterns from an ar- correlation between primer GC content Stoffel fragment produce clearer finger- ray of organisms ranging from bacteria and degree of amplification. ~24~ How- prints with a higher proportion of strong to human. (34'3s) Third, cleavage of tem- ever, some primers produce markedly primary products, are more tolerant of plate DNA with up to five restriction en- fewer amplification products than oth- experimental variables such as primer or donucleases with 4 bp-recognition se- ers, and some produce very few or no enzyme concentration, ~6~ and reveal in- quences prior to amplification did not products with genomic DNA from a wide creased levels of polymorphic DNA.~ss~ decrease the number of amplification range of organisms. ~ss~ The reasons for Several parameters were crucial for products. (34) Destruction of bona fide poor amplification are unclear but may generating reproducible fingerprints. amplicons by cleavage must then permit result from the existence of extremely When compared with RAPD analysis ~24~ amplification of products resulting from rare oligonucleotide sequences as dem- and PCR, DAF requires >10 times more at least single mismatch events at the onstrated in mammals and other organ- primer (3 p.M), equal to higher levels of primer 5' terminus. Fourth, octamers isms (s7-s9) or from interference of the magnesium, and sometimes consider- with single base substitutions at the 5' secondary structure of target site or prod- ably lower template concentrations. r176 end were still able to amplify products uct on the amplification of particular se- Higher primer concentration often re- with defined 8 nucleotide termini by quences. sults in increased primer-template mis- prior amplification. (3s~ Mismatches in matching, favoring the required non- the first 2-3 nucleotides from the 5' end stringent reaction conditions of DAF. PARAM ETERS AFFECTI NG allowed amplification of almost all prod- Consistent fingerprints can be obtained AMPLIFICATION ucts. Mismatches at positions more dis- with relatively low levels of magnesium tant from the 5' terminus failed to ini- Several parameters affect the interaction (1.5-4 mM) for templates or high com- tiate amplification. Fifth, cloning and between single strands, duplexes, hair- plexity and with high levels (4-8 mM) for sequencing of genomic regions that hy- pin loops, and other DNA molecular spe- low-complexity genomes. These require- bridized to several amplification frag- cies formed during MAAP (as depicted by ments vary with ionic components of ments revealed mismatches at the 5' ter- the paradigm of Fig. 1). The use of arbi- the reaction, much as described for minus.(23) trary primers requires adequate discrim- PCR. (64) To avoid possible stoichiometric Addition of nucleotides to the 8 nu- ination of bona fide (i.e., authentic and misrepresentation of amplicons that are cleotide domain does not increase the reproducible) reaction products from rare or amplify with low efficiency, at information content of DNA patterns those of artifactual origin. This discrim- least 1 ng/p.1 or 0.1 ng/p.1 of template significantly. This observation suggests ination must coexist with an inherent DNA from low- or high-complexity ge- that longer primers such as those used in nonstringent reaction environment de- nomes, respectively, must be added. other studies (23-2s) may not be required. termined by the chemistry of the primer. Whereas DNA patterns were consistent The 5' terminus of primers of a length In trying to maintain such fine equilib- over these limits, lower template levels comparable with those used in PCR, rium, the levels of reaction components can result in inconsistent fingerprints, however, may condition fingerprint pat- must be carefully optimized. Frequently, with variant bands corresponding to tern significantly. This is expected be- temperature is varied to favor the forma- weakly amplified products. ~ss'6~

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DAF produces fingerprints from with arbitrary primers, because neither DNA fingerprints that can group and prokaryotic and eukaryotic organisms the primers nor the process is random in classify genomes individually are ob- with minimal experimental variability. nature. tained by screening primers and select- In contrast, artifactual nongenetic varia- Whereas criteria for selection of priming those exhibiting adequate levels of tion in RAPD analysis can be consider- ers vary with the MAAP technique em- polymorphic DNA. In some cases, few able if primer-template concentrations ployed, empirical and theoretical con- primers can accomplish this task easily, and the annealing temperature are not siderations during primer design will as in the analysis of different isolates of carefully optimized. (6s'66) For example, help unveil hidden sequences responsi- Streptococcus uberis ~7~ and cultivars of high and low template concentrations ble for failed or successful amplifica- banana. ~71~ In these studies, DAF and annealing temperatures produced tions. Genomic compositional inhomo- matched bacterial subgroup character- smears. ~3z~ These effects may be respon- geneities occur widely and in different ization and cultivar classification using sible for the failure of RAPD analysis to scales, ranging from mammalian isoch- physiological, biochemical, and molecu- produce profiles with heptamer and oc- ores of high GC content to prokaryotic lar approaches (restriction endonuclease tamer primers known to give complex repeated palindromes. ~s9~ These devia- fingerprinting, 16S ribosomal DNA anal- profiles with DAF. tions from randomness should account ysis, and simple sequence length poly- for some of the departures observed in morphism (SSLP) analysis). In contrast, the calculation of the expected number isolates of Discula destructiva were very Thermal Cycling Parameters of amplification products. ~34~ difficult to separate even after screening Shorter primers require lower annealing many primers. (72) In such cases, other temperatures for adequate amplifica- tailoring strategies had to be used. Diges- tion. However, a pentamer can still pro- APPLICATIONS tion of template DNA with restriction duce DAF products at -55~ r Because endonucleases prior to amplification AFLPs arise from a variety of mecha- these short primer-template duplexes was found to enhance the detection of nisms, including nucleotide substitu- should be unstable at these tempera- polymorphic DNA in fingerprints gener- tions that create or abolish primer sites tures, (67~ the few primer annealing ated with a particular primer. ~73~ Fungal and insertion, deletion, or inversion of a events that occur allow primer extension and plant cultivars that were indistin- priming site or of segments between and accumulate product. Sequence-re- guishable by DAF with several primers priming sites. DNA rearrangements related primers of 7-15 nucleotides in were easily separated in this way. sult in length polymorphisms that some- length failed to amplify DNA at anneal- DAF has been used to characterize a times manifest as presence or absence of ing temperatures of 65~176 Tem- wide range of organisms, ranging from DNA fragments. These DNA polymor- peratures of >80~ were unable to ren- bacteria to human (for review, see refs. phisms constitute identification tools der profiles with any primer, probably 31 and 74). Despite producing simple for genetic typing and mapping. because higher temperatures do not al- patterns, RAPD has been used exten- low adequate primer annealing and ex- sively to fingerprint bacteria ~s2's4'Ts-77~ tension. While DAF profiles tended to and fungi, Cs1'7~-83~ study phylogenetic DNA Fingerprinting, Molecular, simplify with increasing annealing tem- and taxonomical relationships in Taxonomy, and Population Genetics perature, trends in pattern distribution plants, ~84-89~ and establish molecular re- and the boundary of the 8-nucleotide Arbitrary primers can be used to define latedness and genetic variation in ani- primer domain were conserved. The use genetic relationships between organisms mals. (5~176 Moreover, RAPD mark- of "hot start ''(ls'16~ in these experiments in evolutionary, phylogenetic, and taxo- ers were used successfully in population did not alter DNA patterns or product nomical studies. MAAP strategies also and pedigree analysis, ~93-9s~ to test inter- yield. DAF appears very tolerant of an- constitute straightforward identification specific nuclear gene flow in Irises, (96~ nealing, extension, and denaturation tools for general fingerprinting, whether and to predict evolution of androdioecy times; however, other MAAP techniques used in forensic science, clinical patho- in flowering plants. ~97~ Combined with may not an excessive number of temper- gen identification, or plant varietal cer- denaturing gradient-gel electrophoresis, ature cycles should be avoided, both be- tification. Their versatile nature is sug- RAPD detected polymorphisms in cereal cause of long experimental times and gested by how fingerprints can be easily crop species. ~9s'98~ AP-PCR has been used generation of artifacts. tailored to be simple or complex by to study relatedness of Leptospira, ~99~ de- changing amplification components termine parentage in maize, ~1~176identify and primer sequence, length, and num- mouse strains, (1~ and detect somatic Primer Design ber. ~31~ Simple DNA patterns, generally genetic alterations in colorectal tu- The design of primer sequence, even if used for genetic mapping, can be ob- mors.(102) performed using established algo- tained with lower primer or magnesium In cases where the template is com- rithms, (68'69) is not a random process. concentrations in RAPD analyses ~24~ or posed of more than one genome, it is Primers are arbitrary from the moment with shorter primers and higher anneal- possible to differentiate each organism we design their sequence to the inevita- ing temperatures in DAF.~2s'6~ Complex without resorting to physical separation ble selection of those that are informa- patterns, better suited for DNA finger- of the contributing genomes. In the tive and render "useful" DNA polymor- printing applications, can be better pro- study of the Azolla-Anabaena symbiosis, phisms. We should then eliminate the duced with engineered DNA poly- characteristic DAF profiles for the fern or word "random" from our lexicon when merases (truncated or cloned versions) the microsymbiont were generated by al- connected to the amplification of DNA or higher magnesium concentration. ~ss~ tering primer sequence. (1~

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High-resolution Genetic Mapping in lettuce, (~16~ and fruit ripening and This endonuclease-linked MAAP strategy pedicel abscission in tomato. (1~7) can be used efficiently to generate STSs MAAP has provided genetic markers in RAPD markers linked to a particular or sequence-characterized amplified re- biological systems that have not lent locus using near-isogenic lines or BSA gions (SCARs) (119~ linked to genes of in- themselves easily to study. Genetic link- are usually found only after screening terest, for high-resolution linkage map- age analysis in conifers, complicated be- hundreds of arbitrary primers. This lim- ping of specific genomic regions and, cause of the large size of the genome and itation can be overcome by enhancing potentially, for chromosome walking. the difficult generation of F2 segregating the detection of polymorphic DNA. The Usually, MAAP markers possess the populations, has been possible with the high information content of DAF finger- intrinsic disadvantages of dominance, use of arbitrary primers. ~61~ High-density prints and digestion of template DNA low allele number, and lack of homol- maps were constructed with unprece- with restriction endonucleases prior to ogy among related taxa. Sometimes, dented speed in white spruce ~1~ and amplification increase significantly the they target dispersed repetitive DNA se- loblolly pine ~l~ using haploid DNA levels of polymorphism. ~73) The strategy quences, making them useless as hybrid- from megagametophytes. A map of Ara- uses a limited number of primers to gen- ization probes. In some cases, these char- bidopsis thaliana with >250 RAPD mark- erate diagnostic markers that can distin- acteristics devalue their use as genetic ers was created in only 4 person-months guish near-isogenic lines or pooled sam- markers or even as sources of conven- using recombinant inbreds. (~~ Simi- ples in BSA. Using this approach, we tional RFLPs. (62~ Some of these limita- larly, segregation of markers in recombi- differentiated two ethylmethane- tions can be overcome if amplification nant inbred strains allowed genomic sulfonate-induced mutants of soybean products are sequenced and turned into mapping in mouse. ~176 MAAP mapping altered in the nts locus that controls nod- STSs or SCARs. PCR can then be used re- has been also validated in soybean, (24'73~ ule formation. The supernodulating nts liably to link overlapping clones in phys- diploid alfalfa,(~~ ~~ yellow trait segregates as a single recessive Men- ical mapping. (12~ Because DAF can fin- birch, ~176 mouse, and rat. (53'~~ delian locus and is unlinked to two sep- gerprint yeast chromosomes and large arately isolated non-nodulation loci (for genomic DNA fragments isolated by review, see Caetano-Anoll~s and Gress- pulse-field gel electrophoresis ~2~ and Strategies for Directed Mapping: hoff). (~8~ Several DAF markers were can analyze templates composed of Analysis of Near-isogenic Lines, found tightly linked to the nts locus after more than one genome, ~176 MAAP of Bulked Segregant Analysis, and screening only 19 primers (Fig. 3). (73~ cloned DNA can conceivably confirm MAAP of Endonuclease-digested The abundance of linked markers sug- identification of overlaps in yeast artifi- DNA gests that a "hot-spot" of mutagenic ac- cial chromosomes (YACs) and cosmid li- MAAP markers can be linked to specific tivity occurred closed to the nts locus. braries. Certain arbitrary primers should chromosomal regions and can be used to isolate genes of interest without requir- ing a genetic map of the entire genome. RAPD markers located in regions intro- gressed during development of near-isogenic lines were identified and found linked to nematode ~ and Pseudomo- has O13) resistance genes in tomato, or to downy mildew resistance in lettuce. (1~4~ However, near-isogenic lines require ex- tensive backcrossing for their generation and may contain cointrogressed regions of the donor genome that are not linked to the trait in study. Based on a genome pooling RFLP strategy, (l~s) bulked segregant analysis (BSA) (H6~ allows mapping of a gene that segregates in a single population, construction of detailed maps of specific genomic regions, saturation of those regions with markers, or coalescing of unlinked sections of a genetic map. The strategy uses bulked DNA samples from segregating individuals that differ for a FIGURE 3 Detection of DAF markers linked to the supernodulation nts locus. DNA from Glycine soja (accession number PI468.397), soybean cultivar Bragg, supernodulating EMS-induced mu- specific phenotype or genotype. The tant lines nts382 and ntsl007, and 10 representative supernodulating Fz segregants from a cross bulked samples are screened with arbi- between G. soja and ntsl007 were digested with MspI, Hinfl, and TaqI and amplified with primer trary primers, and polymorphic markers GACGTAGG. Whereas digestion separated Bragg from the nts mutants, (7a~ profiles show a 280-bp are identified. Variations of this tech- AFLP (arrowhead) that also differentiates the nts allelic mutants. A double arrowhead indicates 1 nique were originally used to target re- of 21 AFLPs found tightly liked to the nts locus after screening only 18 octamer primers. Sizes gions responsible for disease resistance shown are in kb.

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produce YAC-characteristic fingerprints printing of single cells. Nucleic Acids Res. 20. Hartley, J.L. 1991. Amplification of nu- by amplifying specifically the cloned 16: 10953-10971. cleic acid sequences using oligonucle- DNA away from the common yeast ge- 6. Tautz, D. 1989. Hypervariability of sim- otides of random sequence as primers. nomic DNA background. MAAP would ple sequences as a general source of U.S. Patent No. 5,043,272. polymorphic DNA markers. Nucleic Acids then behave much as AIu-PCR, a tech- 21. Zhang, L., X. Cui, K. Schmitt, R. Hubert, Res. 17: 6463-6471. W. Navidi, and N. Arnheim. 1992. nique used specifically to amplify hu- 7. Litt, M. and J.A. Luty. 1989. A hypervari- Whole genome amplification from a man genomic DNA in YACs. <18~ able microsatellite revealed by in vitro single cell: Implications for genetic anal- amplification of a dinucleotide repeat ysis. Proc. Natl. Acad. Sci. 89: 5847-5851. CONCLUSION within the cardiac muscle actin gene. 22. 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Amplifying DNA with arbitrary oligonucleotide primers.

G Caetano-Anollés

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