Amplifying DNA with Arbitrary Ollgonucleotide Primers

Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Amplifying DNA with Arbitrary Ollgonucleotide Primers 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- 3:85-949 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 PCR Methods and Applications 85 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Review lllllll 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 exami- nation 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 deter- mine 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

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