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Vol. 9, Issue 4, 383-392, April 1999
METHODS
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ABSTRACT |
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 Top
 Abstract
 Introduction
RESULTS
DISCUSSION
METHODS
References
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The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
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INTRODUCTION |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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With conventional cloning methods, linear DNA inserts to be cloned
are generated by either PCR amplification or by the cleaving action of
restriction endonucleases that leave the DNA fragments with blunt ends
or specific overhangs. In a second step, the
corresponding ends of a DNA insert are covalently joined to the
appropriately prepared complementary ends of a plasmid vector by the
action of DNA ligase (Fig. 1A). Here, we present a
new approach to molecular cloning that exploits the unique activity of
a single enzyme, Vaccinia DNA topoisomerase I, to both cleave
and rejoin DNA strands with a high sequence specificity. The enzyme, a
314-amino-acid virus-encoded eukaryotic type I topoisomerase (Shuman
and Moss 1987
), binds to duplex DNA and cleaves the phosphodiester
backbone of one strand at a consensus pentapyrimidine element
5'-(C/T)CCTT in the scissile strand (Shuman and Prescott 1990;
Shuman 1991a,b). In the cleavage reaction, bond energy is conserved by
formation of a covalent adduct between the 3' phosphate of the
incised strand and a tyrosyl residue (Tyr-274) of the protein (Fig.
1B,C). The covalent complex can reclose across the same bond originally
cleaved (as occurs during DNA relaxation) or it can combine with a
heterologous acceptor DNA that has a 5' hydroxyl tail complementary
to that of the adduct, and thereby create a recombinant molecule, as
first described by Shuman (Shuman 1992a,b).
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Topoisomerase I-mediated cloning uses the above reaction to join DNA
fragments containing 5' hydroxyl groups to acceptor plasmid vectors. PCR fragments are well-suited for this topoisomerase-mediated ligation step because they generally have 5' hydroxyl residues from
the primers used for the amplification reaction (Fig. 1D). In fact,
only DNA that has a 5' hydroxyl group can serve as a substrate for
the topoisomerase-mediated ligation and this contributes to the low
rate of vector-only transformants (Shuman 1994). In addition, because
low-melt agarose and slightly elevated temperatures (22-42°C) do
not interfere with topoisomerase I activity, it is possible to purify
desired DNA fragments by electrophoresis through low-melt agarose
followed by excision of the appropriate band. This DNA purification
method is amenable to high-throughput and ensures that only the desired
DNA fragments are included in cloning reactions. These features of
topoisomerase I, as well as the speed of its DNA rejoining activity,
have been exploited to develop a high-throughput cloning technique.
This high-throughput cloning technique serves as the platform in the process for accelerated cloning and expression of open reading frames (ORFs) that is described here. We have performed two feasibility studies to demonstrate the suitability of this process for genome-scale molecular cloning and expression. In one study, we attempted to clone all 6035 ORFs of the yeast Saccharomyces cerevisiae into both the yeast pYES2/GS and the mammalian pcDNA3.1/GS expression vectors, and we then tested the positive-orientation clones for their ability to direct recombinant protein synthesis in yeast and in Chinese hamster ovary (CHO) cells, respectively. In the second feasibility study, we demonstrated the power of this technology for cloning and expressing human cDNAs. In this case, primer sets for 288 human kinases were used to amplify full-length ORFs from cDNA, and these ORFs were then taken through the cloning and expression process. The results are presented below.
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RESULTS |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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High-Throughput Cloning of Yeast ORFs
In this study 6035 ORFs from S. cerevisiae were amplified by PCR and inserted into two separate expression vectors (pYES2/GS and pcDNA3.1/GS; see Fig. 2A,B). The plasmids were tested for insert orientation, and orientation-positive plasmids were expression tested in yeast or CHO cells. There are essentially six phases to this process, which are schematically represented in Figure 2C and outlined below. Process details are given in Methods.
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Phase I
Amplification
Phase IIInsert Purification
when we attempted to clone straight
from the PCR reaction, we obtained an unacceptably high number of
clones that contained primer-dimer PCR products (data not shown). Phase
I and II resulted in the isolation of 5632 ORFs, for an amplification
success rate of ~93%.
Phase IIITopoisomerase I-Mediated Cloning
Phase IVDiagnostic PCR
Phase VPlasmid Preparation
Phase VIExpression Testing
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High-throughput Cloning of Human Kinase ORFs
Next, we analyzed the possibility of utilizing this cloning system,
coupled with an initial high-throughput reverse transcriptase-PCR (RT-PCR) (Saiki et al. 1985; Sambrook et al. 1989) step, to obtain full-length human ORFs and insert them into the pcDNA3.1/GS vector. We
focused on human ORFs that encode kinases, a family of proteins involved in signal transduction. The results described below are summarized in Table 1.
RT-PCR
To assess the feasibility of large-scale RT-PCR amplification of full-length human ORFs, we designed primer sets to amplify 288 full-length human kinases. PolyA+ mRNA was isolated from human fetal heart tissue, converted to first-strand cDNA, and used as template for PCR amplifications primed with the 288 human kinase primer sets (see Methods). A single pass with these 288 primer pairs resulted in RT-PCR generation of 179 products of the predicted size.Topoisomerase I-mediated Cloning, Diagnostic PCR, and Plasmid Preparation
The 179 RT-PCR products were cloned into the pcDNA3.1/GS vector using the protocol described in the previous study. Diagnostic PCR on eight colonies from each of 179 transformations indicated that 140 of the 179 PCR products were cloned in the correct orientation into the expression vector. This represents a 78% success rate for this step. One or two colonies harboring these plasmids were grown overnight in deep-well 96-well blocks and plasmid DNA was prepared (in cases in which diagnostic PCR identified more than one positive-orientation clone, DNA was prepared from two clones and transfectedsee below).
Expression Testing in CHO Cells; Sequence Analysis of Plasmids
The expression plasmids bearing the 140 unique PCR products were transfected into CHO cells in 96-well, deep-well blocks. Cell lysates were made 48 hr after transfection and assayed by Western blot (Fig. 3). Analysis of positive signals and comparison to expected mobilities indicated that 115 of the transfected plasmids directed synthesis of an appropriately sized recombinant protein. So far, sequence data have been obtained for 113 of these 115 constructs: 108 (95%) contained the appropriate kinase and the predicted insert/vector junctions. Four of the incorrect plasmids contained DNA inserts of the correct size but the incorrect identity, and the fifth contained the correct insert, but in the reverse orientation. These five inserts did not contain a stop codon in the same frame as the epitope tag, resulting in expression of a fusion protein of the predicted size, but which was not the correct protein. The overall success rate of the process from RT-PCR through positive expression was 38% (108/288). Generally, in most transfections performed with DNA prepared from two unique clones bearing insert from the same RT-PCR, both DNAs directed expression of the protein of the predicted size (data not shown). This feasibility study (excluding the sequencing) took the equivalent of one person two weeks to complete.
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Success RatesYeast vs. Human ORFs into Vector pcDNA3.1/GS
As mentioned above, the overall success rate for cloning the yeast ORFs into pcDNA3.1/GS, starting with amplification PCR and finishing with expression-positive QC in CHO cells, was 9% for the first pass, and 22% for the second pass. In contrast, the overall success rate for cloning and expressing the human kinases was 38% in a single pass. This single pass also included an RT-PCR step (which was not needed with the yeast ORFs) to acquire the full-length template for cloning. Table 2 summarizes the efficiencies at each step of the process for the yeast clones and the human kinase clones. Possible reasons for the differing success rates are given in the Discussion.
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ORF Length Correlates Negatively to Success Rate of Diagnostic PCR
It is to be expected that success rates for several phases of the high-throughput cloning process will be influenced by the size of the ORF being processed. We have analyzed the data from the first and second pass cloning of yeast ORFs into vector pYES2/GS, as well as data from a smaller-scale topoisomerase I-mediated cloning of yeast ORFs into the same vector (data not shown), and have determined the efficiency with which we were able to identify plasmids containing positive-orientation inserts of a given length. The analysis is limited to phases III and IV of the cloning process, in which a purified yeast ORF (YORF) is mixed with topoisomerase I-adapted vector, the mixture is transformed into bacteria, and diagnostic PCR is performed on eight colonies from each transformation. Figure 4A was produced by sorting YORF PCR products by size into groups of increasing size (250 bp increments). For each group, we divided the number of YORFs for which at least one positive-orientation plasmid was identified by the total number of YORFs in that size group (Fig. 4B). These data clearly indicate that success rate of diagnostic PCR correlates negatively with increasing ORF length. For example, at least one positive-orientation plasmid was obtained for 80% of the 1001- to 1250-bp YORFs taken through phases III and IV, whereas at least one positive-orientation plasmid was obtained for only 22% of the 3751- to 4000-bp YORFs taken though the same phases. Because the data were collected from the diagnostic PCR reactions performed on colonies resulting from 12,284 separate cloning events (total from the two passes and those from the smaller-scale test cloning mentioned above), the statistical significance is high.
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DISCUSSION |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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During and following the first pass with the yeast ORFs, we made several improvements to the cloning and expression process. Most of these changes are process-related and are as follows: (1) low-melt agarose is now prepared fresh, to avoid generation of break-down products that interfere with the normally robust topoisomerase I-based cloning; (2) the amplification and diagnostic PCR primers have been replaced with primers of better design (see Methods); (3) the yeast induction protocol now includes short (3 hr) and long (24 hr) time points, so that yeast clones that express recombinant protein for a short window of time are identified; and (4) CHO cell culturing procedures were altered to ensure that cells were kept at lower, more transfection-competent, passage numbers. Further improvements to the process are the result of the steadily increasing skills of the personnel involved in the study.
These improvements were incorporated into a second pass through the yeast genome, and start-to-finish success rates improved significantly (see Tables 1 and 2). Because the ultimate goal of these projects was to produce reagents for the research community, only plasmids that directed Western blot-detectable synthesis of the correct recombinant protein were judged to be expression-positive. In the case of plasmids made in the pcDNA3.1/GS vector, there are several steps in the expression testing at which expression levels are likely to be reduced due to high-throughput requirements: (1) Miniprep DNA is used for the transfections, and the 96-well transfection format limits the number of cells per transfection to 3 × 105; (2) the cells are all harvested 48 hr after transfection, which might be too long if the recombinant protein is toxic, but too short if the protein accumulates slowly; and (3) transfections are done according to a fixed schedule, making it difficult to ensure that cells are at the optimal density and passage number for transfection. In the case of pYES2/GS-based plasmids, the yeast transformations, selections, and inductions are all performed in 1.4-ml cultures in 96-well blocks, conditions that are well-suited to high-throughput expression testing, but cannot be the optimal expression conditions for each plasmid. Therefore, it is likely that a percentage of both the pYES2/GS- and the pcDNA3.1/GS-based clones do direct recombinant protein synthesis, but at levels below our detection methods. We are currently assessing alternative methods, such as DNA sequencing or more sensitive protein detection techniques, to test the validity of the orientation-positive plasmids that did not direct Western blot-detectable levels of recombinant protein synthesis.
The human kinase study was undertaken after most of the high-throughput cloning and expression techniques had been optimized during the yeast ORF first pass, and this is reflected in the high rate of success of the kinase project (38% from start to finish; see Table 1). This study produced 108 unique pcDNA3.1/GS clones, each proven by Western blot to direct synthesis of the appropriately sized recombinant protein, and each partially sequenced to confirm the insert identity and to ensure that the insert/vector junctions were as predicted. This work also demonstrated that RT-PCR could be used for high-throughput generation of full-length ORFs from human mRNA.
A comparison between the human kinase and YORF projects reveals that
77% of the orientation-positive pcDNA3.1/GS human kinase clones
directed synthesis of the appropriate protein (i.e., were expression-positive), whereas only 15% of the orientation-positive pcDNA3.1/GS YORF clones were expression-positive. Even in our improved
second pass at the yeast genome, only 37% of the orientation-positive pcDNA3.1/GS clones tested expression-positive. The difference in
success rates for yeast ORFs and human ORFs at this stage of the
process might be explained by two fundamental differences between the
amplified yeast ORFs and the amplified human ORFs. First, each
amplified human ORF contains only a Kozak consensus sequence (CACC)
(Kozak 1987) appended upstream of the start ATG, whereas each yeast ORF
contains a palindromic sequence (5'-GCAGTCGTGGAATTCCAGCTGACCACC) appended immediately upstream of its ATG. It is possible that the extra
palindromic sequence in each yeast ORF interferes with transcription
and/or translation of the ORF. A second difference between the human
and yeast ORFs is that the human ORFs were amplified once, with
first-strand cDNA used as template, whereas the yeast ORFs were
PCR-amplified twicethe first time by Research Genetics, using a
250:1 TaqI to PfuI polymerase (nonproofreading
and proofreading polymerases, respectively) mixture and employing
genomic DNA as the template; and a second time by us, in reactions that
used the Research Genetics amplification products as templates and used
a higher-fidelity 50:1 TaqI to PfuI mixture
(Barnes 1992, 1994). Therefore the twice-amplified yeast ORFs may
contain a higher number of nonsense mutations than the human ORF
amplification products. We are currently conducting a variety of
experiments, including the sequencing of positive-orientation clones,
to determine the reasons why some correctly oriented clones do not
direct Western blot-detectable levels of recombinant protein synthesis.
Analysis of the YORF cloning/expression results from the first pass revealed a strong inverse correlation between the length of an ORF and the likelihood of identifying a plasmid containing that ORF in the positive orientation. The stage analyzed encompasses phases III and IV of the cloning process, and these phases consist of three manipulations that might be influenced by YORF length: topoisomerase-mediated ligation of amplified ORFs contained in low-melt agarose plugs, bacterial transformation, and diagnostic PCR. Experiments are under way to determine which of these steps is most adversely affected by clone length. Our analysis also suggests that it may be advantageous to group PCR primers in plates according to the size of the expected amplification product. This will make it possible to pick additional transformants from the plates that contain the longer ORFs, therefore increasing the probability of obtaining orientation-positive clones.
Whereas the above studies used topoisomerase I to create recombinant
plasmids, there exist numerous methods for high-throughput cloning of
PCR products. For example, ligase could have been used to join the PCR
products to expression vectors. This method, however, requires that PCR
products be phosphorylated, either through the use of phosphorylated
primers or by phosphorylation of the PCR product. Also, measures must
be taken to ensure a low rate of vector-alone (containing no insert)
transformants. Vector dephosphorylation is commonly employed, but this
treatment often results in vectors that accept inserts with poor
efficiency. Alternatively, vectors can be created so that the cloning
site is located within a gene that encodes a lethal protein (Bernard et
al. 1994). These vectors cannot give rise to bacterial transformants
unless the lethal gene is interrupted by a cloned DNA. A drawback of
this method is that the lethal gene sequences constrain the sequences
surrounding the cloning site, thereby making it difficult to include
desired elements in the vector (promoters, terminators, epitope tags, etc). Another method to improve ligase-mediated cloning efficiency, TA
cloning (described in Fig. 1A), relies on vectors prepared with single
3' T overhangs to limit vector-alone transformants. This method has
been shown to be very effective for cloning PCR products and could
certainly be adapted to high-throughput applications. Because ligase
activity is reduced by increased temperatures and the presence of
low-melt agarose, however, TA-cloning is not compatible with the
streamlined method of PCR product purification that we used with
topoisomerase I-mediated cloning.
There also exist recombination-mediated cloning strategies that use
yeast (Muhlrad et al. 1992; Oldenburg et al. 1997) or Escherichia
coli cells (Zhang et al. 1998). These methods generally do not
suffer from problems associated with vector-alone transformants. Also,
yeast-based recombination schemes offer a unique advantage in the
generation of yeast expression plasmids because the recombinant plasmids can be functionally tested in the cells in which they were
created. These methods, however, require that the PCR primers each have
30-40 bp of homology to the recombination target, which increases
primer cost by over twofold. In addition, these methods involve
transformation of PCR products, which precludes the use of the low-melt
agarose method for DNA purification. Finally, it is not trivial to
retrieve recombinant plasmids from yeast cells, a necessary step if the
plasmids are to be sequenced or transferred into a different host.
Our studies have demonstrated that topoisomerase I-mediated cloning is a robust method to create recombinant plasmids and that it is particularly well-suited to large-scale cloning efforts. We have also shown that high-throughput RT-PCR can be used in conjunction with this cloning technique to greatly facilitate high-speed cloning and expression of ORFs from organisms whose genomic DNA contains introns.
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METHODS |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Preparation of Topoisomerase-Adapted Vectors
This protocol is used to prepare both the pcDNA3.1/GS and the pYES2/GS vectors. The vector is cut with HindIII, extracted with phenol/chloroform, and ethanol precipitated. TOPO-H (5'P-AGCTCGCCCTTATTCCGATAGTG) and TOPO-4 (5'-AGGGCG) oligos are ligated onto the HindIII-cut vector, the vector is phenol/chloroform extracted, ethanol precipitated, cut again with HindIII to remove re-circularized vector, and again phenol/chloroform extracted and ethanol precipitated. Purified Vaccinia topoisomerase I and TOPO-5 oligonucleotide (5'-CAACACTATCGGAATA) are added to the vector and the mixture is incubated for 15 min at 37°C (buffer is 1× NEB restriction buffer 1; New England Biolabs, Beverly, MA). During this step, the topoisomerase I cleaves after, and remains covalently attached to, the second T in the CCCTT sequence in ligated oligonucleotide TOPO-H. This leaves a vector with topoisomerase I bound to a 3' overhanging T. The reaction is stopped by addition of 1/10 volume of TOPO-10× stop buffer. Free oligonucleotides and unbound topoisomerase I are purified away from the topoisomerase-adapted vector by agarose gel electrophoresis.
High-Throughput Cloning of Yeast ORFs
Phase IPCR Amplification
Phase IIInsert Purification
Phase IIITopoisomerase I-Mediated Cloning, Bacterial
Transformation, and Plating
Phase IVColony Picking and Diagnostic PCR on Bacterial Cells
Phase VPlasmid Preparation
Phase VIExpression Testing
EXPRESSION TESTING OF PCDNA3.1/GS PLASMIDS IN CHO CELLS
In each well of a deep-well 96-well block, a mixture of 24 µg of PerFect Lipids (pFx-6) (Invitrogen, Carlsbad, CA) and 5 µg of plasmid DNA was added to 488 µl of Opti-Mem reduced serum medium (GIBCO Life Technologies, Baltimore, MD). This mixture was shaken for 5 min at room temperature, then 3 × 105 CHO suspension cells (in 500 µl Opti-Mem) were added to each well. The deep-well block was shaken for an additional 5 min and placed in a 37°C humidified incubator. After 4 hr, the cells were pelleted and the medium was replaced with 1.5 ml of CHO-S-SFM medium (GIBCO Life Technologies). The cells were incubated for 42-48 hr at 37°C, then pelleted and lysed in the 96-well blocks, and the lysates were loaded by eight-channel pipettor onto nine-well Bio-Rad 12% Tris-glycine polyacrylamide gels. Five microliters of Novex (San Diego, CA) Sea Blue markers, along with 50 ng of a control protein for the anti-V5/HRP conjugated antibody (Invitrogen, Carlsbad, CA), was loaded into lane one of each gel. Proteins were transferred to Schleicher & Schuell Optitran membrane filters, and the filters probed with the anti-V5 antibody (Invitrogen, Carlsbad, CA). Immunolocalized antibody was detected by incubation with Pierce (Rockford, IL) SuperSignal Ultra chemiluminescence and subsequent exposure to film. Transfected plasmids that directed synthesis of the correctly sized fusion protein were marked as Western positive.EXPRESSION TESTING OF PYES2/GS PLASMIDS IN YEAST CELLS
Approximately 4 µg of each plasmid DNA was transformed into competent INVSc1 S. cerevisiae cells (his3
1 leu2
trp1-289 ura3-52) in a 96-deep-well block [transformation was
performed essentially as described in S.c. EasyComp kit from Invitrogen
(Carlsbad, CA), with the exception that 25 µl of competent cells
were used for each transformation]. The cells were cultured in
selective growth medium (1.3% yeast nitrogen base, 2% glucose, 20 µg/ml histidine, 20 µg/ml tryptophan, and 30 µg/ml leucine)
for 3-4 days at 30°C, then pelleted (3000g, 10 min), and
the medium was replaced with induction medium (1.1% yeast nitrogen
base, 2% galactose, 1% raffinose, 20 µg/ml histidine, 20 µg/ml tryptophan, 30 µg/ml leucine). After overnight induction,
the cells were pelleted, the medium decanted, and each pellet
resuspended in 15 µl 1× DNase buffer [50 mM Tris-Cl at pH 7.4, 5 mM MgCl2, 0.1 mg/ml of DNase Igrade
II (Boehringer-Mannheim, Chicago, IL) 1 mM PMSF, and 5%
glycerol]. Fifteen microliters of 2× sample buffer [0.5
M Tris-Cl at pH 6.8, 20% glycerol, 10% (wt/vol) SDS, 0.1%
Bromophenol Blue, 700 mM 
-mercaptoethanol] was added to
each well, the entire 96-well block was placed in boiling water for 5 min, then the block was placed on ice. Samples were analyzed by Western
blot as described above for the CHO cell lysates. During the yeast ORF
first pass, it was noticed that some plasmids directed recombinant
protein synthesis for a short window of time. To ensure that we
detected protein synthesis in subsequent similar cases, we removed 750 µl of culture from each well of the 96-well block after 3-hr
induction. These cells were pelleted, frozen and saved. Fresh induction
medium (750 µl) was added to each of the wells of the 96-well block
and the induction was continued overnight. These cells were then pooled
with the short-induction cells, and the combined cells were processed
as described above. We have noticed that the expressed recombinant proteins consistently run 2-5 kD high when compared with the Sea Blue
markers. The reason for this is not known, but it could be that the
loading buffer used for the Sea Blue markers is different from our
lysis/loading buffer.
High-Throughput Cloning of Human Kinase ORFs
RT-PCR
Primer pairs were designed to amplify 288 full-length human kinases from the ATG to the last codon before the stop codon. These primers were designed to have a melting temperature of ~60-64°C and each 5' primer included a Kozak sequence (CACC) immediately preceding the ATG to increase translational efficiency (Kozak 1987). Fetal human
heart tissue was obtained from the International Institute for the
Advancement of Medicine (IIAM), Scranton, PA. The Micro-FastTrack 2.0 Kit (Invitrogen, Carlsbad, CA) was used to isolate polyA+ mRNA. The
mRNA was converted to first-strand cDNA using the cDNA Cycle Kit from
Invitrogen (Carlsbad, CA), using the oligo(dT) primer provided and the
protocols suggested. Eight cDNA synthesis reactions were split into the
wells of a 96-well PCR amplification plate, and PCR amplifications were
performed for the 288 human kinase primer sets. Cycling parameters were
1 cycle of 94°C for 4 min; 35 cycles of 94°C for 45 sec, 55°C
for 45 sec, and 72°C for 3 min; followed by 1 cycle of 72°C for 4 min. Each reaction was performed in 50 µl total volume and included
2 units of a 50:1 (unit/unit) mixture of TaqI (Sigma, St.
Louis, MO) and PfuI (Stratagene, San Diego, CA) polymerases,
2.0 µl of 50 mM dNTPs, 2-20 ng first-strand cDNA, and 5 µl of TA 10× PCR buffer (Invitrogen, Carlsbad, CA). Each primer
was added to a final concentration of 0.6 nM. The reaction
products were separated on a 1% low-melt agarose gel and visualized by
ethidium bromide staining. DNA bands that represented correctly sized
amplification products were removed from the rest of the gel using
disposable transfer pipettes and placed into appropriate corresponding
wells of a 96-well microtiter plate. These plugs were subsequently
melted and used as inserts in cloning reactions as described in the
previous section.
Diagnostic PCR on Bacterial Cells
Diagnostic PCR was performed as described above for the yeast ORFs, with the exception that a 5' gene-specific primer was used in conjunction with primer H6STOPREVU for each reaction. In this case, only cells containing the correct ORF in the correct orientation should give amplification products.DNA Sequencing
Each expression-positive human kinase plasmid was sequenced across the cloning junctions and approximately 350 bp into each end of the insert. This was performed on the Licor IR2 system, using labeled primers that flanked the cloning site.Expression Testing
See protocol described for the yeast ORFs.| |
ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health (NIH) grant 1 R01 CA80224-01. We thank Heidi Kijenski and Jim Snook for programming the QIAGEN BioRobot to perform the essential task of colony re-racking. We thank Maria Knoske, Kelly Wynne, Jill Bloom, Sandy Adams, Shannon Hattier, and Josh Uhlig for performing some of the work described in this study. We also thank Karyn Hoeffler for aid in PCR primer design, Cinzia Ellero for help in writing the manuscript, and Doreen Crawford for assistance with the figures.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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FOOTNOTES |
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1 Corresponding author.
E-MAIL john_heyman{at}invitrogen.com; FAX (760) 603-7201.
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REFERENCES |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Received October 26, 1998; accepted in revised form February 23, 1999.
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1 (for DNA that was
negatively or positively supercoiled, respectively).
(C) In the cleavage reaction, bond energy is conserved
by formation of a covalent adduct between the 3' phosphate of the
incised strand and a tyrosyl residue (Tyr-274) of the protein. The
covalent complex can reclose across the same bond originally cleaved or
it can combine with a heterologous acceptor DNA that has a 5'
hydroxyl tail complementary to that of the adduct, and thereby create a
recombinant molecule. (D) Topoisomerase I-mediated cloning
uses the reaction mediated by Vaccinia DNA topoisomerase I to join
PCR-amplified DNA fragments into plasmid vectors. PCR fragments have
5' hydroxyl residues from the primers used for the amplification
reaction, and therefore are an ideal substrate for the topoisomerase
ligation reaction. The topoisomerase I (solid black shape) is shown
linked to the vector through the 3' phosphate (P) of the incised
strand. The PCR product has single 3' A overhangs (A).
1
positive-orientation clones by the total number of YORFs in that size
group.