John M. Butler^§, Christian M. Ruitberg, and Dennis J. Reeder
National Institute of Standards and Technology, Biotechnology Division, Gaithersburg, MD 20899
^§Current address: GeneTrace Systems, Inc., 333 Ravenswood Avenue, Menlo Park, CA 94025

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An internet-accessible database containing information about commonly used short tandem repeat (STR) DNA markers is now available to benefit research and application of STRs to human identity testing. A comprehensive listing of over 500 references focused on STRs are compiled in STRBase. Facts and sequence information on STR systems are included along with population data, common multiplex STR systems, and published PCR primers. In addition, STRBase contains a review of various technologies for resolving STR alleles as well as a summary of validation studies performed on various STR loci. Hyperlinks to paternity testing laboratory web sites and forensic organizations are also a part of this database. STRBase may be accessed via the World Wide Web at http://ibm4.carb.nist.gov:8800/dna/home.htm.

The polymorphic nature of tandemly repeated DNA sequences that are widespread throughout the human genome have made them important genetic markers for gene mapping studies, linkage analysis, and human identity testing (1). While there are literally hundreds of STR systems that have been mapped throughout the human genome (2), only a few dozen STR loci have been investigated for application to human identity testing (3-6). These STR loci are found on almost every chromosome in the genome and may be amplified using a variety of polymerase chain reaction (PCR) primers. Tetranucleotide repeats have been most popular among forensic scientists due to their fidelity in PCR amplification, although some tri- and pentanucleotide repeats are also in use (1,5). Desirable features for STR systems include a high heterozygosity, a regular repeat unit, distinguishable alleles, and capability for robust amplification using PCR (7).

While the use of STRs for genetic mapping and identity testing has become widespread among DNA typing laboratories, there is no single place where information may be found regarding STR systems. The literature on DNA typing by STR analysis is spread out over hundreds of papers spanning the last six or eight years. A busy forensic scientist entering the new world of STR typing could easily feel overwhelmed. In addition, the nomenclature for allele designation often differs among laboratories, making comparison studies difficult at best. One example of variation between laboratories is in the repeat structure nomenclature for the STR locus HUMTH01. The Forensic Science Service designates the repeat structure as TCAT (4,5) while the Promega Corporation lists the repeat as AATG (8). This difference may be rectified by an examination of the GenBank sequence for HUMTH01 (http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank; D00269). TCAT is the first complete repeat on the top strand while AATG is the first full repeat on the bottom strand. Having access to sequence information for STR loci makes them easier to understand and can resolve apparent conflicts.

During the past year, with a goal of making future work with STRs and human identity testing easier, we began work on an STR review paper. We wanted to bring together the abundant literature on the subject in a cohesive fashion. The compilation of material regarding sequence information, observed alleles, and primer sequences quickly grew to a size that no respectable journal would consider publishing nor would anyone have time to read it in its entirety. We also realized that in a field that is developing as rapidly as DNA typing, a dynamic database would have greater value to the scientific and legal community rather than a static document. New information could constantly be entered into a database, whereas a published review paper merely marked the time when it was published and could only represent the past. The internet, and particularly the World Wide Web, with its ability to link information within a document and to be reached by virtually anyone in the world became the focus of our attention. The material which had been gathered to prepare the review paper was translated into hypertext markup language (HTML) using Microsoft^® FrontPage 97–and STRBase was born (Figure 1).

At the heart of STRBase is information describing each commonly used STR DNA marker. These "STR Fact Sheets" contain information on observed alleles (including reported microvariants) with their repeat structure and their PCR product sizes using published primers (Figure 2). Published reports of repeat sequence structures are followed or are converted to a common nomenclature. In almost all cases, we use the top strand from GenBank^® for allele nomenclature and repeat sequence information. Each primer sequence or new reported allele is referenced to a comprehensive STR reference listing (see reference listing section below). Hyperlinks are included to GenBank^® (9) making the entire sequence for the STR locus readily accessible. The number of repeats in the GenBank^® sequence is also noted for each locus. Additionally, a list of population studies and references specific to the STR locus of interest may be reached via hyperlinks. Multiplex sets of STRs (see section below) and commercial sources for allelic ladders are hyperlinked as well. STR Fact Sheets may also be reached through a hyperlinked chromosomal index of identity testing markers (Figure 3). A listing of original papers for each STR locus in also part of STRBase. As of September 1997, STR Fact Sheets are available on STRBase for the following loci: CD4, CSF1PO, D5S818, D7S820, D12S391, D13S317, D16S539, DYS19, F13A1, F13B, FES/FPS, FGA, HPRTB, HUMTH01, LPL, TPOX, and VWA.

Sequence information from GenBank^® (http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank) for commonly used STR loci is accessible through STRBase. Using the accession number for each STR locus, the GenBank^® sequence was downloaded from the internet and then trimmed to include the repeat region and the flanking regions with previously reported primer sequences. In the JPEG sequence files found in STRBase, the repeat region and the published primer sequences are annotated (Figure 4). For example, the top strand repeat in HUMTH01 of TCAT and the bottom strand of AATG are both highlighted (see Fig. 4). The smaller primer set, developed by the Forensic Science Service (4), produces a 170 bp PCR product for allele 9 (the GenBank^® sequence) and the larger primer set, developed by Caskey’s group (1,3), generates a 195 bp amplicon.

It is important to point out that the repeat structures and even the number of repeats may differ depending on whether the top or bottom strand (as found in GenBank^®) is used in the allele designation (Table 1). We recommend using the top strand from GenBank^® for designating the repeat sequence of an STR marker as GenBank^® is publicly available, and this allows for a common nomenclature.

Laboratories expend a great deal of time and effort to conduct studies on allele frequencies for various STR systems with populations that are of interest to their lab. There are over 750 population studies which have been published and now brought together in one place for the first time through STRBase. Most of the studies contain a sample size of greater than 100 samples, which is usually sufficient to make reliable projections about a genotype’s frequency in a larger population (19).

Published population studies have been documented in STRBase by listing the STR system, the population examined, the number of unrelated individuals tested, and the reference. This index of STR population studies should be valuable for locating references that contain useful allele frequencies to aid in calculating matching probabilities for DNA typing cases. In addition to the complete list of 750 population studies, population data has been sorted by STR locus and is available for CD4, CSF1PO, D21S11, DYS19, F13A1, F13B, FES/FPS, FGA, HPRTB, HUMTH01, LPL, SE33, TPOX, and VWA. Unfortunately, copyright laws prohibit including complete allele frequency information from previously published work in STRBase.

Before a new STR system or STR multiplex may be routinely employed in human identity testing, it should be extensively validated to ensure reliability of results. STRBase contains summaries of many of the validation studies which have appeared in the literature as well as a listing of the TWGDAM guidelines for validation of PCR-based DNA typing markers. These materials should help future researchers design their validation studies and allow those who use common STR systems to review what studies have been performed previously.

The constant development of new technologies for DNA analysis makes it difficult for an individual to keep up with or sometimes even understand some of the new methodologies. We have included a brief review of techniques that have been successfully applied to resolving and detecting STR alleles. The established techniques of polyacrylamide gel electrophoresis with silver staining, fluorescent scanning, and automated fluorescent detection systems are discussed along with the emerging methods of capillary electrophoresis, capillary array electrophoresis, microchip CE analysis, and time-of-flight mass spectrometry. Pertinent references in the literature for each technology and hyperlinks to groups working in each area are also included.

PCR relies upon the binding characteristics of the oligonucleotide primers used to define the targeted region of DNA. A great deal of effort is often expended to design primers which bind specifically to the region of interest and which do not form primer dimers. Since the positions of the primers define the size of the PCR products, primers are sometimes redesigned when STR multiplexes are prepared. Different groups have often designed unique primers for the same STR loci. The STR Fact Sheets take this into account by listing each primer set with the subsequent PCR product sizes. In this section, we have broken up the published PCR primers into two groups: the Forensic Science Service and Caskey’s group at Baylor (many of which have been adopted by Promega). It should be remembered that published primers are not necessarily those used by commercial sources for multiplex amplification.

Hyperlinks are also made to organizations involved in DNA typing, commercial sources of instrumentation or DNA testing kits, paternity testing laboratories, electronic journals where STR publications have been found, and other useful DNA databases, such as GenBank^® and the Genome Data Base. Table 2 includes some of the web site addresses which contain information on STR markers or may be of interest to forensic scientists.

References from journals, conference proceedings, and book chapters were gathered and entered into Reference Manager. Over 500 references pertaining to STRs and their application to DNA typing are listed in STRBase. These references come from almost 50 sources including many conference proceedings and several book chapters (TABLE 3).

We have a section of STRBase containing information on Y-chromosome STRs, which may prove useful for rape cases and paternity testing. The known alleles and PCR product sizes for 10 Y-chromosome STRs are listed as described in several papers (10-13). DYS19, DXYS156, DYS287, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393 have 58 reported possible alleles.

Various PCR-based sex-typing assays have been reported in the literature. The most widely used one is amelogenin, which differentiates a 6 bp deletion on the X-chromosome from the Y-chromosome. We have listed primer sequences, PCR product sizes, and references for several sex-typing markers including amelogenin (14,15), the centromeric alphoid repeat (16), and the ZFX/ZFY zinc finger gene (17,18).

Addresses for scientists working with STR markers are available in STRBase with the idea that email links can be made to every scientist doing DNA typing with STRs. Most of these addresses were obtained from the correspondence addresses listed in published papers. We invite others to add their name, phone number, and e-mail address to this portion of STRBase to aid cooperation of DNA typing laboratories around the world.

The short tandem repeat DNA database is available to the general public through the World Wide Web: http://ibm4.carb.nist.gov:8800/dna/home.htm (Figure 1). When using information from STRBase, please cite this paper and the date which the information was gathered from STRBase.

The information contained in STRBase is taken from published works on short tandem repeats used for DNA typing purposes. The literature is regularly searched for new publications and updates to STRBase are made from time-to-time. Comments on the database, suggestions for further improvements, or submissions should be sent to Dr. Dennis J. Reeder, attn: STRBase, National Institute of Standards and Technology, Biotechnology Division, Building 222 Room A353, Gaithersburg, MD 20899 or dennis.reeder@nist.gov.