XbaI R6181

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	After amplification, the PCR products were digested by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique separately with the restriction enzymes, with recognition of the following sequence: 5′...CAG ↓ CTG...3′ and 5′...T ↓ CTAGA...3′, respectively for Pvull and Xbal in a final volume of 20 μl at 37 °C for 16 h, containing 6 U Pvull and 5 U XbaI (Promega), 2 μl enzyme buffer, 0.2 μl BSA (bovine serum albumin), and 17.3 μl PCR product.	Fragment analysis was performed in 1% agarose gel stained with ethidium bromide, and the bands were separated and photographed under ultraviolet light. The PCR and RFLP conditions utilized followed the protocol of Souza et al. [16, 17].

Protocol tips
After amplification, the PCR products were digested by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique separately with the restriction enzymes, with recognition of the following sequence: 5′...CAG ↓ CTG...3′ and 5′...T ↓ CTAGA...3′, respectively for Pvull and Xbal in a final volume of 20 μl at 37 °C for 16 h, containing 6 U Pvull and 5 U XbaI (Promega), 2 μl enzyme buffer, 0.2 μl BSA (bovine serum albumin), and 17.3 μl PCR product.
Downstream tips
Fragment analysis was performed in 1% agarose gel stained with ethidium bromide, and the bands were separated and photographed under ultraviolet light. The PCR and RFLP conditions utilized followed the protocol of Souza et al. [16, 17].

Publication protocol

After amplification, the PCR products were digested by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique separately with the restriction enzymes, with recognition of the following sequence: 5′...CAG ↓ CTG...3′ and 5′...T ↓ CTAGA...3′, respectively for Pvull and Xbal in a final volume of 20 μl at 37 °C for 16 h, containing 6 U Pvull and 5 U XbaI (Promega), 2 μl enzyme buffer, 0.2 μl BSA (bovine serum albumin), and 17.3 μl PCR product. Fragment analysis was performed in 1% agarose gel stained with ethidium bromide, and the bands were separated and photographed under ultraviolet light. The PCR and RFLP conditions utilized followed the protocol of Souza et al. [16, 17]. The restriction enzymes and the base pair (bp) sizes for the normal and variant alleles are summarized in Table 1. We excluded three participants of control group due to problems with the blood collection. The final number is 101 girls in this group. We included the database in the supplemental files (Additional file 1).

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