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Short tandem repeat (STR) genotyping methods are widely used for human identity testing applications, including forensic DNA analysis. Samples of DNA containing the length-variant STR alleles are typically separated and genotyped by comparison to an allelic ladder. Here, we describe a newly devised library of cloned STR alleles. The library covers alleles X and Y for the sex-determining locus Amelogenin and 259 other alleles for 22 autosomal STR loci (TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D6S1043, D12S391, Penta E, D19S433, D11S4463, D17S974, D3S4529 and D12ATA63). New primers were designed for all these loci to construct recombinant plasmids so that the library retains core repeat elements of STR as well as 5′- and 3′-flanking sequences of ∼500 base pairs. Since amplicons of commercial STR genotyping kits and systems developed in laboratories are usually distributed from 50 to <500 base pairs, this library could provide universal templates for allelic ladder preparation. We prepared three different sets of allelic ladders for this locus TH01 and an updated phiên bản of an allelic ladder for the DNATyper®19 multiplex system using these plasmids to confirm the suitability of the library as a good source for allelic ladder preparation. Importantly, the authenticity of each construct was confirmed by bidirectional nucleotide sequencing and we report the repeat structures of the 259 STR alleles. The sequencing results showed all repeat structures we obtained for TPOX, CSF1PO, D7S820, TH01, D16S539, D18S51 and Penta E were the same as reported. However, we identified 102 unreported repeat structures from the other 15 STR loci, supplementing our current knowledge of repeat structures and leading to further understanding of these widely used loci.
Keywords

Forensic science; Short tandem repeat (STR); Allelic ladder; Recombinant plasmid; Repeat structure
1. Introduction
During the past two decades, short tandem repeat (STR)
genotyping has emerged as the dominant technique for human
identity determination and paternity testing [1–4]. Allelic ladders,
which serve as standards in STR analysis, are necessary for
adjusting for different sizing measurements obtained from
different instruments and under different conditions used by
various laboratories, which makes allelic ladders an indispensable
component of commercial kits and newly developed STR analysis
systems.
Allelic ladders are prepared on the basis of the polymerase
chain reaction (PCR) for which the templates can be obtained by
three optional strategies. The first strategy is to cut gels after
polyacrylamide gel electrophoresis (PAGE) to collect separated
fragments of target alleles [5]. The DNA template thus prepared is
not suitable for long-term storage because it is inclined to degrade.
Another disadvantage of this strategy is the amount of DNA
dissolved from gels is usually very limited. Once the entire DNA
template is used, the demanding procedures of PAGE and gel
cutting have to be repeated. Additionally, the concentration and
purity of prepared DNA templates from different batches of
experiments can vary significantly. The second strategy is based on
plasmid construction. Each allele is amplified by PCR and inserted
into plasmids that can be transformed easily into Escherichia coli
[6]. Thus, large amounts of genetically engineered plasmids
bearing STR fragments, which are ideal PCR templates for allelic
* Corresponding authors. Tel.: +86 10 66269184; fax: +86 10 63267051.
E-mail addresses: [email protected] (L. Wang), [email protected]
(X.-C. Zhao), [email protected] (J. Ye).

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