Direct cloning of a long restriction fragment aided with a jumping clone.

Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.     

New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin

Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.     

Method for cloning restriction fragments containing the termini of BAC inserts

We have developed a method to isolate the termini of BAC clones. The method is based on the two unique NotI sites located approximately 300 bp on either side of the EcoRI cloning site of the BAC vector pECS-BAC4. Our strategy includes the following steps: (i) generation of Southern blots with BAC clones digested with NotI and a second restriction enzyme; (ii) identification of the termini attached to the NotI/EcoRI fragment of the BAC vector via hybridization with a probe derived from sequences located between one NotI site (left or right arm) and the cloning site; (iii) ligation of the doubly digested BAC clone (NotI and the selected second restriction enzyme) with an equally doubly digested cloning plasmid vector; and (iv) confirmation of the clone as a terminus. This strategy has allowed us to begin the construction of a contig near a common bean gene that controls resistance to a group of potyviruses.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

基因克隆的常用方法介绍

为能快速、准确地克隆出有意义的基因,本文介绍了目前常用的一些基因克隆方法,如差异显示ＰＣＲ、抑制性差减杂交、ＲＡＰ－ＰＣＲ、代表性差异显示、酵母双杂交系统、ｃＤＮＡ直接捕捉法等;并对这些方法作了简要的评价,以利于大家选择适合自己的方法。     

Technique for cloning and sequencing the ends of bacterial artificial chromosome inserts

Bacterial artificial chromosome (BAC) libraries are an important tool for positional cloning, gene analysis and physical mapping. During studies using BAC clones, it is often necessary to organize them into contiguous sequences (contigs). To finalize, join and extend the contigs, both cloning and sequencing of the ends of the inserts are required. Here, we describe a low-cost, accessible, fast and powerful method for the routine isolation of BAC ends. This method allows the isolation of 20 BAC clone ends in one day. The analysis of the ends reveals fragment sizes compatible with sequencing, and the structure of these clones allows the sequencing of both ends using the same plasmid. Moreover, long end fragments can be sequenced in both directions.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

抑制性消减杂交在生物基因克隆中的应用

抑制性消减杂交(SSH)是抑制PCR与消减杂交技术相结合,能对未知序列差异表达基因克隆的一种方法。该方法具有速度快、效率高、假阳性率低、目的序列富集程度高、实验结果复杂程度低等特点。本主要介绍其基本原理、操作过程、优缺点、在生物基因克隆中的应用,并对其应用前景进行了分析。     

Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing

Whilst cloning and sequencing techniques are used ubiquitously for the identification of novel DNA sequences, the necessity to determine a consensus sequence means that this can be both labour‐intensive and costly. Here we describe a rapid and cost effective method of using denaturing gradient gel electrophoresis (DGGE) for the analysis of large numbers of clones prior to sequencing. This procedure allows for the selection of specific clones, eliminates the need to sequence multiple copies of the same clone, and reduces the likelihood of sequencing recombinant PCR product.     

Direct Cloning and Sequencing of Bacterial Artificial Chromosome (BAC) Insert Ends Based on Double Digestion

Conventional digestion and ligation was developed into a novel and efficient approach for directly cloning and sequencing the two ends of bacterial artificial chromosome (BAC) clone inserts. Most BAC vectors have two Not I sites. This end isolation method is based on double digestion of the BAC clone DNA with Not I and any blunt-end restriction enzyme for which there is not a restriction site located within the small fragment (containing the cloning site) between the two Not I sites on the BAC vector. Digestion is followed by ligation of the double-digested mixture with a suitable plasmid vector. The pBeloBAC11 and pBlueScriptII SK vectors were used in the present study. The two ends of the BAC insert can be amplified and sequenced with three specific primers, i.e., amplification of the left end with the pBeloBAC11 LF1 and pBlueScriptII KS primers, and the right end with the pBeloBAC11 RR4 and KS primers. They may be directly recovered by transformation if the end fragments are used as probes. More significantly, this simple strategy generally can be applied to any BAC vector with any cloning site.