Hong Yan, Chuah Aaron
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604.
BMC Bioinformatics. 2003 Feb 25;4:7. doi: 10.1186/1471-2105-4-7.
Amplified fragment length polymorphism (AFLP) is a PCR-based technique that involves restriction of genomic DNA followed by ligation of adaptors to the fragments generated and selective PCR amplification of a subset of these fragments. The amplified fragments are separated on a sequencing gel and visualized by autoradiography or fluorescent sequencing equipment. AFLP allows high-resolution genotyping but the lack of a format for databasing and comparison of AFLP fingerprint profiles limits its wider applications in profiling large numbers of biological samples.
A scheme is described to represent a DNA fingerprint profile with a nucleotide sequence-like format in which the information line contains the minimal necessary details to interpret an AFLP DNA fingerprint profile. They include technique used, information on restriction enzymes, primer combination, biological source for DNA materials, fragment sizing and annotation. The bodylines contain information on size and relative intensity of DNA fragments by a string of defined alphabets or symbols. Algorithms for normalizing raw data, binning of fragments and comparing AFLP DNA fingerprint profiles are described. Firstly, the peak heights are normalized against their average and then represented by five symbols according to their relative intensities. Secondly, a binning algorithm based loosely on common springs and rubber bands is applied, which positions sequence fragments into their best possible integer approximations. A BLAST-like reward-penalty concept is used to compare AFLP fingerprint profiles by matching peaks using two metrics: score and percentage of similarity. A software package was developed based on our scheme and proposed algorithms. Example of use this software is given in evaluating novelty of a new tropical orchid cultivar by comparing its AFLP fingerprint profile against those of related commercial cultivars in a database.
AFLP DNA fingerprint profiles can be databased and compared effectively with software developed based on our scheme and algorithms. It will facilitate wider use of this DNA fingerprinting technique in areas such as forensic study, intellectual property protection for biological materials and biodiversity management. Moreover, the same concepts can be applied to databasing and comparing DNA fingerprint profiles obtained with other DNA fingerprint techniques.
扩增片段长度多态性（AFLP）是一种基于聚合酶链式反应（PCR）的技术，该技术包括对基因组DNA进行酶切，然后将接头连接到产生的片段上，并对这些片段的一个子集进行选择性PCR扩增。扩增后的片段在测序胶上进行分离，并通过放射自显影或荧光测序设备进行可视化。AFLP可实现高分辨率基因分型，但缺乏用于AFLP指纹图谱数据库构建和比较的格式，这限制了其在大量生物样品分析中的更广泛应用。
本文描述了一种方案，该方案以类似核苷酸序列的格式来表示DNA指纹图谱，其中信息行包含解释AFLP DNA指纹图谱所需的最少必要细节。这些细节包括所使用的技术、限制性内切酶信息、引物组合、DNA材料的生物来源、片段大小测定和注释。主体行通过一串定义的字母或符号包含DNA片段大小和相对强度的信息。文中描述了用于原始数据归一化、片段分箱以及比较AFLP DNA指纹图谱的算法。首先，将峰高相对于其平均值进行归一化，然后根据其相对强度用五个符号表示。其次，应用一种基于普通弹簧和橡皮筋的松散分箱算法，将序列片段定位到其最佳可能的整数近似值。使用类似BLAST的奖励 - 惩罚概念，通过使用两个指标（得分和相似度百分比）匹配峰来比较AFLP指纹图谱。基于我们的方案和所提出的算法开发了一个软件包。通过将新热带兰花品种的AFLP指纹图谱与数据库中相关商业品种的指纹图谱进行比较，给出了使用该软件评估新热带兰花品种新颖性的示例。
利用基于我们的方案和算法开发的软件，可以有效地将AFLP DNA指纹图谱构建数据库并进行比较。这将有助于该DNA指纹技术在法医学研究、生物材料知识产权保护和生物多样性管理等领域的更广泛应用。此外，相同的概念可应用于构建使用其他DNA指纹技术获得的DNA指纹图谱的数据库并进行比较。
