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        【共享】端粒长度的定量测量

        丁香园论坛

        1880
        端粒长度的测量方法,是通过实时荧光定量PCR进行的,希望对做端粒的同行有所帮助!
        文件613k,无法上传,贴前言来共享吧。
        INTRODUCTION
        The traditional method of measuring telomere length in
        samples of total human genomic DNA determines a mean
        terminal restriction fragment (TRF) length (1). The method
        requires large amounts of DNA (0.5–5 μg/individual) and time
        (3–5 days). Furthermore, the relative mean TRF lengths of
        individuals can vary by as much as 5% depending on the
        particular restriction enzymes used, suggesting the existence of
        subtelomeric restriction site polymorphisms and/or subtelomeric
        length polymorphisms that may confound the identification of
        primary factors accounting for inter-individual variation in the
        mean length of the true telomeric repeat sequence. More
        recently, methods have been developed that allow multiple
        samples to be compared for their relative content of just the
        telomeric hexamer repeat itself (2–5). However, none of these
        methods is as simple and as amenable to rapid high throughput
        processing of large numbers of samples as the method
        presented below.
        Our strategy for determining relative telomere lengths by
        quantitative PCR was to measure, for each DNA sample, the
        factor by which the sample differed from a reference DNA
        sample in its ratio of telomere repeat copy number to single
        copy gene copy number. This ratio should be proportional to
        the average telomere length. The quantity of telomere repeats
        in each experimental sample was measured as the level of dilution
        of an arbitrarily chosen reference DNA sample that would
        make the experimental and reference samples equivalent with
        regard to the number of cycles of PCR needed to generate a
        given amount of telomere PCR product during the exponential
        phase of PCR amplification. Similarly, the relative quantity of
        the single copy gene in each experimental sample was
        expressed as the level of dilution of the reference DNA sample
        needed to match it to the experimental sample with regard to
        the number of cycles of PCR needed to generate a given
        amount of single copy gene PCR product during the exponential
        phase of the PCR. For each experimental sample the ratio
        of these dilution factors is the relative telomere to single copy
        gene (T/S) ratio. Thus T/S = 1 when the unknown DNA is
        identical to the reference DNA in its ratio of telomere repeat
        copy number to single copy gene copy number. The reference
        DNA sample (to which all of the experimental samples in a
        given study are compared) can be from a single individual or it
        can be a pooled sample from multiple individuals. The T/S
        ratio of one individual relative to the T/S ratio of another
        should correspond to the relative telomere lengths of their DNA.
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