手動ポリメラーゼ連鎖反応法によるカイコの雌雄判別  [in Japanese] Sex determination of the silkworm, Bombyx mori, by manual polymerase chain reaction.  [in Japanese]

Access this Article

Search this Article

Author(s)

Abstract

<p>先行研究から,カイコのW染色体(♀)を特異的に同定できる11のRAPD(増幅断片多型DNA)マーカーが見出された.その中から, W-Yukemuri-SとW-Bonsaiと名付けられたマーカーに着目し,サーマルサイクラー機器を用いて,PCR法(ポリメラーゼ連鎖反応)によるこれらマーカーの増幅条件を決定した:(1)DNA抽出と鋳型DNA量-カイコ後部糸腺からDNAを上手く分離することができ,そのDNAを反応液当たり2.5 ng以上/μLを加えて,鋳型とした.(2)反応の繰り返し回数と反応温度の範囲一反応温度は, 92-96℃で30秒間, 56-61℃で30秒間, 68-73℃で1分間であり,この温度サイクルを30回繰り返した.これら条件下でのPCR産物をアガロース電気泳動で分離すると,単一の雌特異的マーカーバンドが現れた.</p><p>手動PCR法を確立するために,3タイプの水槽-(1)ホットプレート付スターラーに置いた1Lビーカー, (2)スターラーに置いた温度制御器付恒温水槽, (3)温度制御器と噴流ポンプ式撹拌器を備えた恒温水槽-を用いた.これらの水槽の温度は,設定温度の1.5℃以内の範囲で制御することができ,これら水槽にサンプルを順次浮かべる操作を30回繰り返した.その結果,サーマルサイクラー機器を用いたPCRと全く同じ増幅産物が得られ,雌特異的マーカーであるW-Yukemuri-SとW-Bonsaiが単一バンドとして検出された.これらの結果から,手動PCR法によりカイコの雌雄判別ができることが明らかになった.高校生物の実験教材として導入した場合の有用性を4視点から考察した.</p>

<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>

Journal

  • Japanese Journal of Biological Education

    Japanese Journal of Biological Education 56(1), 29-35, 2015

    The Society of Biological Science Education of Japan

Codes

  • NII Article ID (NAID)
    130007719586
  • NII NACSIS-CAT ID (NCID)
    AN0012957X
  • Text Lang
    JPN
  • ISSN
    0287-119X
  • NDL Article ID
    026831857
  • NDL Call No.
    Z18-53
  • Data Source
    NDL  J-STAGE 
Page Top