• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Anchoring a Defined Sequence to the 5 Ends of mRNAs: The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries from a Few Cells

        互联网

        520
        Among numerous applications, the polymerase chain reaction (PCR) (1 ,2 ) provides a convenient means to clone 5′ ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5′ end of the first-strand cDNAs (corresponding to the 3′ end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5′ end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3′ end of mRNAs. Several strategies have been devised to tag the 3′ end of the ss-cDNAs (corresponding to the 55′ end of the mRNAs). We (3 ) and others have described strategies based on the addition of a homopolymeric dG (4 ,5 ) or dA (6 ,7 ) tail using terminal deoxyribonucleotide transferase (TdT) (“anchor-PCR” [4 ]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3 ). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35′ end of ss-cDNAs. This strategy is referred to as SLIC for single-strand ligation to ss-c DNA (8 ).
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序