Hetero-stagger cloning: Efficient and rapid cloning of PCR products
Hetero-stagger cloning: Efficient and rapid cloning of PCR products
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DOI：
10.1093/nar/24.12.2458
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发表时间：
1996-06-15
影响因子：
14.9
通讯作者：
Liu, ZJ
中科院分区：
文献类型：
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作者：
Liu, ZJ
已开发出多种方法来克隆PCR产物，包括钝端克隆(1)、限制性内切(2)、连接非连接非连接克隆(3)、尿嘧啶脱氧核糖基酶(UDG)处理含尿嘧啶的脱氧寡核苷酸(4、5)和TA克隆(6-8)。聚合酶链式反应产物的钝端克隆通常需要处理聚合酶链式反应产物来打磨末端(9)。即使有了治疗方法，钝端克隆也是低效的。提高克隆效率的关键因素是创造具有凝聚力的相容末端。除了钝端克隆，目前各种可用的PCR克隆方法在如何创建粘性末端方面有所不同。在限制性内切酶削减策略中，将限制性内切酶切入到聚合酶链式反应的引物中，并在聚合酶链式反应后通过限制性内切酶酶切产生粘性末端。这一过程涉及两个缺点：(I)限制位点的额外碱基增加了成本，并导致了二聚体的形成；(Ii)分子末端的限制性内切酶功能不佳(2)。在连接非依赖克隆和UDG克隆策略中，分别用T4 DNA聚合酶或尿嘧啶DNA糖基酶处理PCR产物，产生一个12个碱基的悬垂。这些程序效率很高，但主要的缺点是载体制备过程中涉及的复杂问题，以及在聚合酶链式反应引物组(11)上额外增加24个碱基的费用。TA克隆策略利用Taq DNA聚合酶(9)的末端延伸酶活性，在PCR产物的3‘端增加一个额外的碱基，这与模板无关，但是序列特异性的。如果片段上的3‘核苷酸是G，则添加单个G，但如果3’核苷酸是C、T或A，则添加A，当3‘核苷酸是A(10)时，加成效率非常低。因此，TA克隆只能克隆带有扩展A的分子。最重要的是，TA克隆对于用于PCR扩增的较新的校对DNA聚合酶(如PFU或PwO耐热DNA聚合酶(11))并不有用。在本文中，我提出了一种新的PCR克隆方法，称为异交错PCR克隆(图1)。该方法是基于通过制备不同长度的相关引物可以获得不同长度的相关PCR产物的事实。这些相关的聚合酶链式反应产物混合、热变性和退火化形成异源双链，从而产生具有粘性末端的分子。这些具有粘性末端的分子可以很容易地克隆到相容的载体中(图1)。用Taq聚合酶和校对耐热酶扩增的PCR产物可以用这种方法克隆。将三个额外的碱基GGG添加到一半定制的聚合酶链式反应引物的5‘端。另一半的引物是由目标序列确定的，不需要额外的GGG。建立了GGG-上引物+下引物对的PCR反应和上引物+GGG-下引物的扩增反应两种方法。这两个反应的聚合酶链式反应产物是相同的，除了每个反应都含有三个额外的
A variety of methods have been developed for cloning PCR products, including blunt-end cloning (1), restriction cut back (2), ligation-independent cloning (3), uracil DNA–glycosylase (UDG) treatment of uracil-containing deoxyoligonucleotide primers (4, 5) and TA cloning (6–8). Blunt-end cloning of PCR products often requires treatment of PCR products to polish the ends (9). Even with treatments, blunt end cloning is inefficient. The key element in improving cloning efficiencies has been the creation of cohesive compatible ends. Other than blunt end cloning, various current available PCR cloning methods differ in how the cohesive ends are created. In the restriction cut back strategy, restriction sites are incorporated in the PCR primers and the cohesive ends are created by restriction digestion after PCR. This procedure involves two drawbacks:(i) extra bases for restriction sites increases the cost and leads to primer dimer formation; and (ii) restriction enzymes function poorly at the ends of the molecules (2). In both the ligation-independent cloning and the UDG cloning strategy, a 12 base overhang is created by treatment of the PCR products with T4 DNA polymerase or uracil DNA–glycosylase respectively. These procedures are highly efficient, but the main disadvantages are complications involved in vector preparation and the added expense of 24 extra bases to the PCR primer set (11). The TA cloning strategy utilizes the terminal extendase activity of Taq DNA polymerase (9) adding an extra base to the 3′ of the PCR products, which is template-independent, but is sequence-specific. A single G is added if the 3′ nucleotide on the fragment is a G, but an A is added if the 3′ nucleotide is a C, T or A, with very low efficiency of additions when the 3′ nucleotide is an A (10). As a result, TA cloning can only clone molecules with extended A. Most importantly, TA cloning is not useful with the newer proof-reading DNA polymerases for PCR amplifications such as Pfu or Pwo thermostable DNA polymerases (11).In this communication, I present a novel method for PCR cloning termed ‘hetero-stagger PCR cloning’(Fig. 1). The procedure is based on the fact that related PCR products of different lengths can be obtained by preparing related primers of different lengths. These related PCR products are mixed, heat-denatured, and annealed to form heteroduplex thereby molecules with cohesive ends can be generated. These molecules with cohesive ends can be readily cloned into compatible vectors (Fig. 1). PCR products amplified by using Taq polymerase as well as by using proof-reading thermostable enzymes can be cloned by this procedure. Three extra bases, GGG, are added to half of the customized PCR primers at the very 5′ ends. The other half of the primers is made as determined by the target sequences without extra GGG. Two PCR reactions are set up: one PCR reaction with primer pair of GGG-upper primer plus lower primer, and the other PCR reaction with upper primer plus GGG-lower primer. The PCR products of the two reactions are identical except that each one harbors three extra