CN102725406A - 核酸洗脱的改进 - Google Patents

核酸洗脱的改进 Download PDF

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CN102725406A
CN102725406A CN201080059859XA CN201080059859A CN102725406A CN 102725406 A CN102725406 A CN 102725406A CN 201080059859X A CN201080059859X A CN 201080059859XA CN 201080059859 A CN201080059859 A CN 201080059859A CN 102725406 A CN102725406 A CN 102725406A
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A.F.佩奇
B.O.帕克
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Globegroup Life Technology Consulting America Co ltd
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Abstract

本发明涉及将遗传物质（特别是DNA）储存于固体基质上，所述遗传物质在加入固体基质之前已纯化。更特别地，本发明涉及储存纯化DNA的固体基质，该基质已用含有植物多糖菊粉的溶液进行处理。本发明的一个优点在于可将提高量的DNA储存于本发明的固体基质。

Description

核酸洗脱的改进
技术领域
本发明涉及将遗传物质（特别是DNA）储存于固体基质上，所述遗传物质在加入固体基质之前已被纯化。
发明背景
核酸（特别是DNA）的储存，通常是于溶液中在4℃下冷藏至多数天或在-20℃或更低的温度下冷藏更长的时间来实现的。这种方法对于静态储存是昂贵的且占空间。当核酸样本需要运输且样本经常在干冰中运输时，这种方法就表现出更大的问题了。
Burgoyne (WO90/03959)描述了一种方法，在该方法中，生物样本（通常是血液）可被加到固体基质上，该固体基质结合了裂解细胞的试剂。被释放出来的DNA保留在所述固体基质上。这些样本可以在室温下长时间储存。
US 5,939,259和WO03/016552描述了其中与固体基质缔合的DNA可被洗脱下来用于进一步研究的技术。然而在很多情况下，对加于固体基质上的纯化DNA的回收导致约40%的DNA回收，其如通过定量实时PCR的方法所测定。显然需要一种简便的方法，该方法能够在室温下长时间储存DNA而且具有更高的加入DNA的回收率。
发明概述
令人惊讶地观察到，向固体基质加入菊粉（一种存在于植物中的多糖），大大地提高了可从该固体基质洗脱的加入DNA的百分率。当将纯化DNA加入固体基质时这尤其明显。
附图的简要说明
图1显示了菊粉的结构。
发明详述
将多种化学药品加入到固体基质中以研究它们对从固体基质中回收的DNA的收率的影响。特别地，称为FTA Elute (Whatman)的固体基质经证实特别可用于本发明的实施中。然而预期的是，其它类型的固体基质同样可与本发明一起使用。许多固体基质是基于纤维素的。将这些固体基质用稀释于2M异硫氰酸胍的测试试剂的溶液处理。很多化学药品对回收的DNA的收率有很小作用或没有作用。浓度约10%的聚乙二醇(PEG)在回收DNA的量方面有小量的提高。PEG是乙二醇亚单位的长链聚合物。以多种分子量制备PEG，分子量通过每分子中亚单位的平均数来定义。评价了分子量为400、1000和3350的聚合物。观察到PEG 1000产生最佳的加入DNA的回收率结果（数据未显示）且被应用于余下的检测中，但被称为PEG。在PEG的浓度约为25%时，结果在试验间不同；这可能提示了在这些浓度下，固体基质的包被过程存在着小的不一致性。
已经发现，在固体基质经植物多糖菊粉处理后，观察到由固体基质上洗脱下来的DNA的量的提高。发现在固体基质中加入浓度高达20%的菊粉，将固体基质中回收的DNA的收率从没有加入菊粉时的25-40%提高到80%。有迹象表明，除了加入菊粉外还加入10%的PEG，将回收的DNA的收率提高到接近85%。
菊粉是存在于多种植物中的天然存在多糖。它的结构如图1所示。预期的是，菊粉的简单修饰（例如酯化）是可能的，并且仍实现加入的纯化DNA的洗脱的提高。
可将纯化DNA在缓冲液中加入经菊粉处理的固体基质中，所述缓冲液为核酸化学中常规使用的。所用的缓冲液中还可以含有多达10%的PEG 。所述DNA在加入固体基质之前可以通过多种标准实验室技术纯化。
一个重要的考量是回收DNA的收率的提高随时间(即在室温下延长储存)得以保持。已经发现DNA可以增加的收率回收达至少23天。预期的是，这种增加的收率会在甚至更长的储存时间内出现。室温通常是约20℃-25℃，典型为20℃。
实施例
提供本实施例仅用于说明的目的，不应解释为对由随附权利要求所限定的本发明的限制。
实施例1 基质化学修饰
固体基质为来自Whatman的FTA Elute 903。
1）制备4 M的硫氰酸胍贮备液并用不同浓度的测试试剂稀释至2 M。
2)将基质903(2 ¼" x 2 ¼")放入含有硫氰酸胍/测试试剂混合物的盘内，轻轻搅动10秒钟。
3)将基质在金属支架上使用两个吹风机(Simply Basic DS-727)干燥10分钟；一个吹风机置于基质上方30°角15cm处，另一个吹风机以30°角置于基质下方25cm处，使得两个吹风机和基质成一直线。使基质进一步在没有气流的情况下于21 ± 2℃下过夜干燥。
4)基质于干燥器内在室温下储存备用。
实施例2　DNA加入测试固体基质
将人DNA (Roche)以160ng/μl 的浓度点样加在测试固体基质上。通常使用预先打孔的5mm直径的基质圆盘。将圆盘在室温下干燥至少3小时。
实施例3　DNA从固体基质上的洗脱
1)将每个干燥圆盘置于1.5 ml的无菌microfuge管内，然后用500 µl的蒸馏水通过脉冲涡流洗涤3次，共持续5秒钟。
2)将圆盘转移至含有100 µl蒸馏水的0.5 ml的microfuge管内，确保圆盘被完全浸没。
3)将microfuge管置于加热块内，在98℃下加热30分钟。
4)将microfuge管通过脉冲涡流60秒后进行简短地离心。
5)将洗出液转移到新的0.5ml管内，弃去圆盘。洗出液在4℃下储存直至定量。
实施例4　洗出液中DNA的定量
洗出液中DNA的定量通过QPCR使用7900HT热循环仪（Applied Biosystems）进行。反应使用RNA酶P测定和TAQMAN® Universal PCR Master Mix (Applied Biosystems)建立。使用用于实验的相同RocheDNA的10 ng/µl到0.01 ng/µl的系列稀释液绘制四点标准曲线。早期的QPCR定量在96孔板中进行并人工设置。随着液体处理机器人的引入和确认，后来的定量使用384孔板进行。96孔板和384孔板两者都是经过验证的并且亦针对彼此进行测试以检查方法之间的一致性。
结果
结果（表1）显示了与仅FTA Elute相比，用菊粉或PEG浸渍的基质确实导致DNA回收率增加。除此之外，使用含有10% PEG的点样缓冲液引起DNA收率的额外提高（当使用FTA Elute+ 20%菊粉时，DNA的收率为85%）
Figure 201080059859X100002DEST_PATH_IMAGE001
表1：加入浸渍有附加化学药品的FTA Elute基质的DNA (1 µg)的回收百分率。点样缓冲液刚好在加至预先打孔的5mm直径的圆盘之前与DNA混合。对每个条目，n=4。如1.1节所述干燥圆盘和洗脱DNA。
含有20%菊粉的基质显示加入的纯化DNA的最高回收百分比。由于PEG亦鉴定为一种可能的基质浸渍化学的添加剂，所以也制备浸渍有20%菊粉和10% PEG的组合的FTA Elute用于进一步的研究。当与修饰基质化学结合使用时证实含有10% PEG的点样缓冲液进一步提高收率。
还获得了其中加入纯化DNA的圆盘于室温下在干燥器中储存的实验的结果。该结果显示当将基质用菊粉处理时，在DNA洗脱之前可储存圆盘至少23天且具有相近的提高的DNA回收率。
结果还显示测试基质中加入和回收的DNA的量可以低至1ng且高至1μg (所测的最大值)，并仍观察到菊粉处理的提高的效果。
应该理解的是，关于任一个实施方式所述的任意特征可单独使用，或与其它所述特征结合使用，还可与任何其它实施方式或者任何其它实施方式的任意组合的一个或多个特征结合使用。此外，在不背离本发明的范围的情况下也可应用以上未描述的等同物和修改，本发明的范围由随附权利要求限定。

Claims (6)

1. 一种适用于储存纯化DNA的固体基质，所述基质已用含有菊粉的溶液处理。
2. 如权利要求1所述的固体基质，所述基质也用PEG处理。
3. 如权利要求1或2所述的固体基质，其中菊粉处理在高达20%的浓度下进行。
4. 如权利要求1-3所述的固体基质，所述固体基质也含有高达10%的PEG。
5. 一种提高自固体基质洗脱的纯化DNA的收率的方法，所述方法通过在加入所述核酸之前用含有菊粉的溶液处理固体基质来进行。
6. 如权利要求5所述的方法，其中所述固体基质也用PEG处理或者将所述DNA加入含有PEG的缓冲液中。
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