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https://www.abbs.info ISSN 0582-9879 |
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Short Communication |
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[1]. 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)[8],
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”[9],
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)[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)[10]. 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[11].
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 protected]