ABSTRACT
Tandemly repeated DNA sequences generated from single synthetic oligonucleotide
monomers are useful for many purposes. With conventional ligation procedures low yields and random
orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and
random incorporation of linkers containing restriction sites during ligation.
Ligation products are amplified by PCR using the linker oligonucleotides as
primers. Restriction digestion of the PCR products generate multimer
distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and
spacing can be directly used for subcloning or other applications without further treatment.
Since concatenation of DNA elements increases the affinity for transcriptional
factors (
1
,
2
), tandemly repeated DNA sequences are used for a variety of purposes, including functional characterization of transactivating factors (
3
-
5
), purification of transcriptional factors by DNA affinity column chromatography (
1
), Southwestern blot analysis and expression library screening (
6
). The generation of repeated sequences is based on the direct ligation of
synthetic monomers. Since relatively high monomer concentrations are involved
in these reactions, the phosphorylation and ligation reactions are inefficient
and a majority of the ligation products are short oligomers. The small yields
of longer oligomers makes their manipulation difficult and the subcloning
efficiency of long oligomers is characteristically low. Moreover, the end
products of these reactions are random structures containing tandem and
inverted repeats. To avoid these problems, we developed a ligation-PCR protocol to generate long DNA fragments containing numerous tandem
repeats. With this methodology the length, orientation and spacing of the
repeat units may be precisely manipulated.
The protocol utilizes double-stranded `core' monomers containing any desired motif (e.g., enhancer
element), and linkers containing desired restriction sites. Both
oligonucleotides contain specific overhangs to control orientation during
ligation. Ligation is performed with certain ratios of core monomers to linkers
to control the repeat number. Ligation products are size-selected, PCR amplified using single-stranded linkers as primers, and restricted to generate DNA
fragments carrying sticky ends suitable for ligation without further
modification.
We have successfully subcloned six individual enhansons from the chorionic
somatomammatropin gene enhancer (
7
) as tandem repeats. Pairs of complementary oligonucleotides containing 5'-GG-3' and 5'-CC-3' overhangs and various
enhanson sequences were synthesized (e.g., IR1: 5'-AGGATGTTTTCTAAACGAT
GG
, 5'-ATCGTTTAGAAAACATCCT
CC
). The 19 bp linkers also contain 5'-GG/CC overhangs and
Sal
I
-Pst
I-
Bgl
II sites (5'-CGTCGACTGCAGATCTC
GG
, 5'-GAGATCTGCAGTCGACG
CC
). Single-stranded oligonucleotides (2 [mu]g) were phosphorylated separately in 8 [mu]l reactions (37oC * 90 min) containing 1* phosphorylation buffer, 1 mM ATP and 8 U
polynucleotide kinase (Promega). For annealing, each pair of single-stranded oligonucleotides was combined, heated to 90oC and slowly cooled to room temperature. After brief centrifugation
to collect water condensed on the sides of the tubes, 2 [mu]l linkers were added to the phosphorylated monomers (monomer:linker ratio =
10:1). Ligation was initiated by adding 2 [mu]l 10* ligation buffer and 1 [mu]l (3 U) T4 DNA ligase (Promega) directly to 18 [mu]l of annealed oligonucleotides. After incubation at room temperature for 4 h, half of the
ligation product was resolved on a 2% agarose gel. The majority of the ligation products are dimers and trimers and DNA bands representing larger
products are barely visible (Fig.
1
A). Gel slices containing products of ~600 bp were subjected to one freeze-thaw cycle. After centrifugation (14 000
g
for 5 min), 2 [mu]l of the supernatant (2% of the total volume) was used as a PCR template.
The PCR mixture consisted of 10 [mu]l 10*
Taq
Extender buffer (Strategene), 2 [mu]l 10 mM dNTPs, 0.2 [mu]g of each linker as primers in a volume of 98 [mu]l. After denaturation (94oC for 5 min), 1 [mu]l (5 U) of
Taq
polymerase (Boeringer Mainheim) and 1 [mu]l (5 U) PCR Extender (Strategene) were added and subjected to 35 cycles
according to the following protocol: denaturation at 93oC for 1 min, annealing at 56oC for 1 min, extension at 72oC for 3 min, and terminal extension at 72oC for 10 min. PCR products were extracted once with phenol-chloroform, ethanol precipitated and redissolved in
50 [mu]l water.
REFERENCES
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