ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(3): 365-368 CN 31-1300/Q
Direct Cloning of the Unknown Flanking DNA Fragments from a Large Insert without Restriction Mapping
SONG Bao-Liang1, QI Wei1,2, LI Bo-Liang1*
( 1State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China;
2Department of Biological Science and Technology, Nanjing University, Nanjing 210093, China )
Abstract A simple technique for direct cloning of the target DNA fragments from a large insert according to its adjacent known sequence is described here. In this new subcloning method, a large DNA insert is digested and ligated with a linearized plasmid vector to construct a subclone library that is subjected to screening. The bacterial clones in this library are individually picked, grown in a 96-well plate, and then pooled across the rows or columns. Target clones are obtained from the ordered separate pools by PCR-screening with a set of primers, one specific for the adjacent known sequence and the other serving as "anchor primer" specific for the vector sequence. This direct subcloning procedure was efficiently demonstrated by cloning a specific DNA region from a large insert within 2 days without mapping the starting DNA or isolating the digested DNA fragment.
Key words subclone; screening; walking; ACAT
Large-insert clones such as P1, BAC and PAC usually contain the inserted DNA fragments longer than 100 kilobases. Working with these clones, we found it was very often for the researchers to clone and sequence an interested region whose adjacent sequence was known, for example isolating a promoter region whose down stream sequence could be acquired from its cDNA. To obtain the target fragment, the researchers usually do a lot of digestion and electrophoresis to get the physical map of the DNA insert. Then, the starting DNA was digested by appropriate restriction endonucleases, the target DNA fragment was isolated and inserted into a plasmid vector. This traditional procedure is often effective, but has obvious drawbacks. First, mapping a large DNA insert is time-consuming, expensive and tedious work since many enzyme digestions are needed. Moreover, it is sometimes difficult to isolate the target fragment after a large DNA insert is digested because many fragments with similar sizes cannot be exactly determined just by running agarose gel.
So far, numerous other techniques[2-7], including "ligation anchored PCR" (LA-PCR), have been developed for cloning or/and sequencing a target region from a large DNA insert. LA-PCR involves generating primer sites by ligating an adaptor to the starting DNA, amplifying the target region by using one anchor primer and another primer specific for the known sequence, and cloning the PCR product into a plasmid vector. Although elegant, this technique is mainly limited by the high non-specific background. Some improves, such as "suppression PCR" , are developed to increase the specificity of LA-PCR. But this method isn't wildly used because special DNA anchor needed.
Here we describe a direct subcloning method, including construction of subclone library and screening for the target clone by PCR. In this procedure, a set of primers including one specific for the known sequence and the other serving as “anchor primer” specific for the vector sequence were used for PCR-screening the subclone library (Fig.1). The adjacent known sequence was amplified to identify the positive clones. As one of the experimental samples using this subcloning method, we described the cloning of the target DNA region from P1 774 clone containing human genomic DNA encoding acyl-CoA: cholesterol acyltransferase-1 (ACAT-1).
Fig.1 Schematic diagram of the procedure for direct subcloning
The subclone library was constructed with RE1 and RE2 digestion, ligated with pBSK+ (Stratagene) as described in the “Materials and Methods”. PCR was used to screen the subclone library. The PCR amplification was carried out with oligonucleotide primers specific for a known sequence within the large clone DNA and for a vector sequence, respectively. The words in parenthesis show the clone, primer, and endonucleases used as described in the “Materials and Methods”.
1 Materials and Methods
1.1 Materials All restriction enzymes and agarose were from Premega. Taq DNA polymerase and dNTPs were from Sino-American Biotech (Shanghai, China). P1 774 was from Genome Systems. The oligonucleotides were synthesized with an automated DNA synthesizer in Shanghai Institute of Biochemistry.
1.2 Constructing a subclone library P1 774 clone containing human ACAT-1 genomic DNA was prepared according to the instructions provided by the manufacturer (Genome Sys). 3 mg of P1 774 DNA were digested by KpnI and EcoRV at 37 ℃ for 1 h, then treated by CIAP for 30 min, extracted with phenol∶chloroform (1∶1), precipitated with ethanol and dissolved in TE (pH 8.0). 0.5 mg of vector plasmid pBluscriptII+ (pBSK+) (Stratagene) was linearized by the same set of endonucleases, separated by electrophoresis and purified from agarose gel by Prep-A-Gene kit (Bio-Rad). Ligation reaction between P1 774-digested fragments and linearized pBSK+ was carried out at room temperature for more than 1 h using T4 DNA ligase (Premega). 1 μl of the ligated mixture was transformed into Escherichia coli strain XL1-Blue by electroporation. The transformed bacteria were plated onto the LB/agar plates containing the ampicilin/IPTG/X-Gal. The resulting white colonies were randomly picked into the 8×8 matrix ( 64 wells total, 200 ml LB/well) in a 96-well U-bottom multiwell plate with one clone per well, and cultured at 37 ℃ for overnight without shaking.
1.3 Screening of the subclone library by PCR The overnight cultured bacteria from eight wells across a row or eight wells down a column were pooled using a multiwell pipet (30 ml/well), and mixed well. The matrix of 64 wells was therefore reduced to 16 pools, which were used directly for PCR analysis as described below. The overnight-cultured bacteria in 96-well plate and the pooled bacteria were stored at 4 ℃.
The PCR primer specific for the ACAT-1 upstream region is Upp12 (5′-TGGCCTCAAGTG-ATCTGCC-3′). The primer specific for vector sequ-ence is a universal primer M13-20 primer (5′-GT-AAAACGACGGCCAGT-3′). The PCR conditions were as follows: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 10 mmol/L of each Upp12 and M13-20 primer, 200 mmol/L of each dNTP, 1.5 mmol/L MgCl2, 1 u Taq DNA polymerase (Sino-American Biotech Co.) and 1 ml of the pooled bacteria as source of DNA template. At the same time, a negative control using water as template and a positive control in which 1 ml of the ligation reaction mixture was used for the source of template were performed. The amplification was carried out in a DNA Thermal Cycler (MJ Research), which included 35 cycles of reaction with 30 s denaturing at 94 ℃, 30 s annealing at 55 ℃ and 1 min extension at 72 ℃, followed by a 5 min extension at 72 ℃. PCR products were electrophoresed through a 1.5% agarose/TBE gel and visualized with ethidium bromide.
2 Results and Discussion
The overall strategy for this subcloning method is diagrammed in Fig.1. The steps are explained in details in the “Materials and Methods” section and are described briefly as follows. The large DNA insert is digested with two restriction enzymes, RE1 that digests at the known region and RE2 that can be any other restriction enzyme. In case that chimeric DNA was produced in the following ligation reaction, the DNA fragments in the digested mixture are dephosphorylated by CIAP. Then, these DNA fragments were ligated with the linearized vectors, the ligated mixture was transformed into bacteria and the resulting clones are picked into the 96-well plate and cultured overnight. Finally, these clones in the plate were pooled across the rows or columns and screened by PCR. It should be emphasized that the known region adjoining to the interested unknown DNA fragment is amplified during PCR screening.
In the experiment described here, we attempt to clone the flanking region of the human ACAT-1 gene from a genomic DNA clone, termed P1 774. In the known sequences, there is a site for restriction enzyme KpnI that serves as RE1. Another restriction enzyme we randomly selected is EcoRV that serves as RE2. The primers Upp12 specific for a known region and M13-20 serving as “anchor primer” specific for vector sequence were used. Pools were made as Fig.1 and screened by PCR. Then the PCR products were separated by agarose gel electrophoresis. Ethidium bromide staining of the gel revealed that pools of row 4 and column F, as well as positive control, yielded a expected 868 bp band, while all other pools were negative. Thus, the positive clone in a single well (4F) was identified as containing the target DNA fragment adjoining to the known region screened by PCR (Fig.2). Further analysis and sequencing show that it is the target clone containing about 7 kb insert. In a separate experiment, we cloned an about 2.0 kb downstream fragment of human ACAT-2 gene from a large DNA insert of lambda clone.
Fig.2 PCR screening of pooled subclone library for the 5'-flanking region of human ACAT-1 gene
The subclones of the constructed library from P1 774 clone DNA containing the human ACAT-1 genomic DNA were randomly picked and cultured in the 96-well plate with one clone/well in an 8×8 matrix. Pools from columns or rows were screened by PCR. Panel A and B, ethidium bromide staining of PCR products. The templates for each reaction were: 1-8, pools of rows; Nc (negative control), no template; Pc (positive control), 1 ml ligation reaction mixture; A-H, pools of columns. M, DNA marker (Sino-American Biotech Co.).
This method for direct cloning the unknown flanking DNA obviously has a number of advantages. First, no restriction map of the large DNA insert is required, thus this method obviates the labor on mapping a large DNA insert. Second, this procedure enables the target DNA fragment to be cloned directly into the vector without isolating it by electrophoresis. The mutations caused by thermostable DNA polymerases will not be present in the screened positive clone since PCR product is not used for cloning but only for identification. Finally, a known sequence in the large DNA insert can be used to design appropriate primers to screen subclones containing either upstream or downstream fragments by using this method. Step by step, the whole insert can be subcloned into plasmids and sequenced in a short period of time. We have succeeded in doing a lot of subcloning experiments with P1 clone and lambda clone by using this method, while the other large-insert clone DNAs such as BAC, cosmid or PAC can be also subcloned with the same procedure.
In conclusion, the procedure should be applicable to many types of large DNA insert clones in which sufficient sequence information exists for selecting appropriate restriction enzyme site and designing a primer. In many cases, particularly when conventional ordered subcloning methods require doing a lot of work on restriction mapping and target fragments isolation. The method here simplifies subcloning, saves both labor and time, and therefore makes subclone experiments more convenient and cost-effective.
Acknowledgements We thank our colleagues YANG Xin-Ying, YANG Jin-Bo, MA Han-Hui, YANG Li, YAO Wei and ZHANG Nian-Yi， for their helpful discussion.
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Received: November 21, 2001 Accepted: November 30, 2001
This work was supported by the National Natural Science Foundation of China (No.39425005) and Shanghai Science and Technology Commission (No.97XD14022). The nucleotide sequence reported in this paper has been submitted to the GenBank under accession No.AY040205
*Corresponding author: Tel, 86-21-64747035; Fax, 86-21-64338357; e-mail, email@example.com