Sex determination of the silkworm, Bombyx mori, by manual polymerase chain reaction.

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  • 手動ポリメラーゼ連鎖反応法によるカイコの雌雄判別
  • シュドウ ポリメラーゼ レンサ ハンノウホウ ニ ヨル カイコ ノ シユウ ハンベツ

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Abstract

<p>A previous study showed 11 female-specific RAPD (random amplified polymorphic DNA) markers for identifying W chromosome (♀) of the silkworm, Bombyx mori. Out of the markers, we selected two markers named W-Yukemuri-S and W-Bonsai, and amplified by polymerase chain reaction (PCR) using a thermal cycler (machine PCR). The PCR conditions were determined as follows: (1) DNA extraction and template DNA amount - Genomic DNA was successfully isolated from the posterior silk gland of the 5th larva, which was used as a template DNA at the concentration over 2.5 ng/μL reaction mixture, (2) the number of thermal cycle and the range of reaction temperature at each step- A series of 30 thermal cycles was carried out with each cycle consisting of 3 discrete temperature steps at 92-96℃ for 30 sec, 56-61℃ for 30 sec and 68-73℃ for 1 min. In these conditions, a female-specific PCR product (W-Yukemuri-S) was appeared as a distinct single band after agaroseelectrophoresis. For establishment of manual PCR, three types of water bath were used: (1) 1L beaker on a hot plate stirrer, (2) temperature-controlled water bath on a stirrer, and (3) temperature-controlled water bath with a circulator. The temperatures of these water baths were well regulated within the range of 1.5℃ of the setting temperature. Using these water baths set at different temperatures (94, 60, 70℃), manual PCR was made by a series of 30 repeated temperature changes. The products by manual PCR were identical to those of machine PCR, meaning that two female specific markers, W-Yukemuri-Sand W-Bonsai, are amplified by manual PCR and the silkworm sex can be determined as a result. The incorporation of this experiment into high school biology laboratory is discussed from 4 different angles.</p>

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