Introduction

D1S80 (pMCT118, GenBank accession number D28507) is a variable number of tandem repeat locus located on chromosome 1p35-36 with a repeat unit of 16 bp (Nakamura et al. 1988; Kasai et al. 1990). The D1S80 locus is very useful for human identification because of its high polymorphism, especially in the Japanese population (Rand et al. 1992; Sugiyama et al. 1993; TWGFDM 1995). Baechtel et al. (1993) described an allelic ladder marker that consisted of alleles 16–37 and 41 of the locus, but they were not sequenced. Applied Biosystems (Foster City, Calif., USA) supplied the AmpliFLP D1S80 Allelic Ladder consisting of alleles 14 and 16–41 of the locus, but they only sequenced alleles 14, 16, 18, and 24 in the allelic ladder marker. In addition, Applied Biosystems stopped supplying the AmpliFLP D1S80 Allelic Ladder in 2003. When using the AmpliFLP D1S80 Allelic Ladder, we were not able to type alleles larger than allele 41. However, the alleles larger than allele 41 have been observed with a frequency of about 1% in the Japanese population (TWGFDM 1995). In order to type them, an allelic ladder marker that contains some alleles larger than the allele 41 is required. Here, we constructed a new allelic ladder marker consisting of alleles 13–44 of the D1S80 locus and confirmed that all the alleles had the correct length corresponding to the allele number by sequencing.

Materials and methods

Blood samples were collected from 23 Japanese individuals that were heterozygous at the D1S80 locus. Appropriate informed consent was obtained from each individual. DNA samples were purified from their blood samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA samples were amplified with D1S80 primers (Kasai et al. 1990), and each allele was separated on a 2% agarose gel or by denaturing high-performance liquid chromatography using a WAVE system (Transgenomic, Omaha, Neb., USA). Each separated D1S80 allele was cloned to the pT7Blue T-Vector (Novagen, Madison, Wis., USA).

The plasmid DNAs containing the alleles 13–44 of the D1S80 locus were mixed and amplified with D1S80 primers to construct an allelic ladder marker. The PCR was performed using 0.14–1.2 fg of each plasmid DNA in a 100-μl reaction volume with a TaKaRa PCR Thermal Cycler Dice (Takara Bio, Otsu, Japan). The PCR reaction mixture contained 1× Ex Taq buffer (2 mM Mg2+ Plus, Takara Bio), 0.2 mM dNTPs, 0.05 U/μl TaKaRa Ex Taq HS (Takara Bio), and 1.4 μM of each of the D1S80 primers. The thermal cycle was performed at 95°C for 1 min, followed by 27 cycles at 94°C for 1 min, 65°C for 1 min, and 70°C for 1 min. A final extension was performed at 70°C for 10 min.

All the plasmids containing the alleles 13–44 were sequenced. Four hundred nanograms of the plasmid DNA was sequenced with the T7 promoter primer, with the M13-21 primer, and with the Forward2 primer (5′-CAGCC CAAGG AAGAC AGACC-3′) reported by Watanabe and Shimizu (2002). The sequence reaction with the T7 promoter primer was performed at 95°C for 5 min, followed by 25 cycles at 95°C for 30 s, 46°C for 10 s, and 60°C for 4 min. The sequence reaction with M13-21 was performed at 95°C for 5 min, followed by 25 cycles at 95°C for 30 s, and 60°C for 4 min. The sequence reaction mixture of the T7 promoter primer and of the M13-21 primer contained 5% dimethyl sulfoxide and 5% glycerol. The sequencing reaction with the Forward2 primer was performed with the method described by Watanabe and Shimizu (2002).

Results and discussion

An allelic ladder marker consisting of the alleles 13–44 for use in D1S80 typing was constructed (Fig. 1). The forward sequences were obtained from all the plasmids with the T7 promoter primer and the Forward2 primer, and the reverse sequences were obtained with the M13-21 primer. From the sequence data of the alleles 13–44, 13 types of repeat units were obtained, and the repeat units have been reported previously (Fig. 2, Duncan et al. 1997; Arakura et al. 1998; Liu et al. 1999; Watanabe and Shimizu 2002). The repeat units 2–13 consisted of 16 bp, and their sequences were designated as RMRRA CCACH RGVAA G. The first four repeats and the last three repeats were conserved expect for the allele 21. The allele 21 appears to contain a nucleotide substitution in the 19th repeat. However, we do not know whether the nucleotide substitution occurred in the PCR amplification before the cloning or was derived from an individual who provided the allele. All the alleles used in this study contained the same 5′ and 3′ franking region sequences that were similar to the sequence reported by Kasai et al. (1990). The lengths of all the alleles in this study were calculated using the following formula reported by Sekiguchi et al. (1993): 116 bp (5′ franking region) + 14 bp (first repeat) + 16(n−1) bp (second repeat through last repeat) + 32 bp (3′ franking region) = 146+16n bp, where n is the allele number. The sequencing result revealed that all the alleles in our allelic ladder marker had the correct length corresponding to the allele number, and we think that this marker can be used for more effectively typing D1S80. Takara Bio now supplies the TaKaRa D1S80 Allelic Ladder using the method decribed in this study.

Fig. 1a, b
figure 1

A new sequenced allelic ladder marker for D1S80 typing. a Lane 1 A new allelic ladder marker consisting of alleles 13–44, lane 2 AmpliFLP D1S80 Allelic Ladder (Applied Biosystems) consisting of alleles 14 and 16–41. b Lanes 1, 4, 7, 10 A new allelic ladder marker, lanes 2, 3, 5, 6, 8, 9 PCR products of blood samples amplified with D1S80 primers (lanes 2, 29–44; lanes 3, 16–31; lanes 5, 24–36; lanes 6, 30–42; lanes 8, 18–27; lanes 9, 30–43)

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Fig. 2
figure 2

Sequence structures of alleles 13–44 of the D1S80 locus

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