Caetano-Anollés G, Gresshoff P M
University of Tennessee, Knoxville, USA.
Biotechniques. 1996 Jun;20(6):1044-8, 1050, 1052 passim. doi: 10.2144/96206rr02.
DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.
利用在其3'末端带有任意“核心”序列的微型发夹进行DNA扩增指纹分析（DAF），用于对多种模板进行指纹分析，包括PCR产物和全基因组，以在种间和种内水平建立植物分类群之间的遗传关系，并鉴定密切相关的真菌分离株和植物种质。未观察到任意核心序列、微型发夹结构的稳定性与DAF效率之间存在相关性。还使用了具有短任意核心的微型发夹引物以及与微卫星中存在的简单序列重复互补的引物，从扩增图谱中生成任意特征（ASAP）。ASAP策略是一种两步扩增程序，在每个指纹分析阶段使用至少一种引物。在仔细优化扩增参数（如引物和模板浓度）后，ASAP能够可重复地扩增DAF产物（代表约10 - 15 kb的序列）。为了产生独特的指纹，必须避免引物序列与DAF产物末端部分互补。这使得寡聚物能够在核酸筛选中组合使用，基于有限数量的引物序列进行大量的ASAP指纹分析反应。微型发夹引物和ASAP分析显著提高了多态性DNA的检测率，区分了密切相关的狗牙根（Cynodon）品种，并在大豆（Glycine max）超结瘤（耐硝酸盐共生）位点的混合分离群体分析中检测到了推定连锁的标记。
