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Focus: PCR Cloning
Cloning Differential Display-PCR Products with pGEM®-T Easy Vector System
PCR
products were cloned using the pGEM®-T Easy Vector System (Cat.# A1360)
and the LigaFast™ Rapid DNA Ligation System (Cat.# M8221). |
By Rosalia Sirchia, Ph.D, Valentina Ciacciofera, and Claudio
Luparello, Ph.D.
Dipartimento di Biologia Cellulare e dello Svliuppo, Universita di Palermo, Italy
Introduction
The identification of differentially expressed genes to investigate a specific
developmental system or the genetic changes associated with a given pathology is one of
the most commonly pursued tasks in molecular and cellular biology. Diverse techniques
allow the study of the modulation of gene expression including differential display
(DD)-PCR(a). DD-PCR permits the isolation of cDNA
reverse transcribed from mRNA, allowing comparison of gene expression of differentially
treated samples. The cDNA fragments are cloned and identified by sequencing and homology
searches. The differential expression of the cDNA can then be confirmed using more
stringent techniques for the evaluation of steady-state mRNA such as Northern blots and
semi-quantitative PCR.
We are currently analyzing gene expression in cultured breast cancer cells in response
to microenvironmental modulators such as parathyroid hormone-related peptide (PTHrP),
which is known to affect cell proliferation and invasivity (3,4). Using mRNA
DD-electrophoretograms and re-amplifying and purifying selected cDNA fragments (5), we
have obtained a number of DNA preparations suitable for sequencing and subsequent
identification of the parental transcript.
Here we report the successful use of LigaFast™ Rapid DNA Ligation System* (Cat.# M8821)
and pGEM®-T Easy Vector System (Cat.# A1360)
for cloning PCR products. This procedure yields enough recombinant plasmid for sequence
analysis.
Results and Discussion
To identify genes selectively regulated in a breast cancer cell line after treatment
with a midregion fragment of PTHrP, we isolated cDNA fragments by DD-PCR (5). The cDNA
fragments were ligated into pGEM®-T Easy Vector. When working with DD-PCR
insert-containing vectors, we found that plating 300–500µl of transformed bacterial
cells generated a higher number of colonies with a consequent higher percentage of white
colonies. Additionally, the 6:1 insert:plasmid ratio yielded a 50% increase of colony
number. On the other hand, when the control DNA fragment was used for transformation, an
insert:plasmid ratio of 3:1 and 100µl of transformation culture gave optimal results,
(positive control yielded more than 90% white colonies). The number of colonies grown in
the background control was negligible.
Figure 1 shows the
electrophoretic analysis of recombinant plasmids purified from the cell lysates; we were
able to obtain 1-5µg of recombinant plasmid from 5ml cultures, an amount suitable for
automated sequencing. Figure 2
shows the restriction analysis of recombinant plasmids digested with EcoR I,
whose recognition site closely flanks the insertion site. We found that after enzymatic
treatment, DNA fragments corresponded in size to those of the DD-PCR bands plus the
remnant region of the polylinker.
We compared the colony-forming efficiency of three different bacterial strains, JM109,
HB101 and XL1 Blue. JM109 cells yielded 20% more colonies, both blue and white, than the
other two cell types. We have also successfully cloned DNA fragments extracted and
purified from agarose gel bands.
In conclusion, we can recommend the pGEM®-T Easy Vector System, in
conjunction with LigaFast™ Rapid DNA Ligation System, as a versatile tool for the
quick cloning of DD-PCR products and the production of recombinant plasmids for subsequent
sequencing.
Materials and Methods
Preparation of cDNA fragments: We amplified cDNA fragments (180-750bp)
obtained from DD-PCR using PCR Master Mix (Cat.# M7501).
Annealing temperatures and cycling profiles were empirically determined for each reaction.
For the A-tailing reaction, the elongation time of the last cycle was prolonged to 20
minutes. We determined the purity and the amount of the PCR products by agarose gel
electrophoresis.
Ligation and transformation reactions: The ligation reaction was set
up using pGEM®-T Easy vector and LigaFast™ Rapid DNA Ligation System
using a PCR product:vector molar ratio of 3:1 or 6:1. Positive and background controls
were prepared in parallel as recommended by the manufacturer (6). The reactions were
incubated overnight at 4°C, after which they were quickly centrifuged and 2µl added to
50µl of either JM109, HB101 (Cat.# L2011) or
XL1 Blue (kind gift of Prof. Ida Albanese and Mario La Farina) competent cells.
Transformation was performed according to manufacturer’s instructions (6) onto
LB/Ampicillin/IPTG/X-Gal plates.
Plasmid extraction: White bacterial colonies were picked and grown
overnight at 37°C with agitation in 5ml Luria broth supplemented with 25µl Ampicillin
(100µg/ml). Polypropylene tubes were used for colony incubation. Recombinant plasmids
were purified using a commercial miniprep kit and quantitated by gel electrophoresis.
Insertion of the PCR product of interest into the plasmid was verified by EcoR I
digestion of 200–400ng of recombinant plasmid and analysis by agarose gel
electrophoresis.
Acknowledgments
Work partially supported by Italian MURST (Cofin and ex 60%).
References
- Sokolov, B.P. and Prockop, D.J. (1994) A rapid and simple PCR-based method of isolation
of cDNAs from differentially-expressed genes. Nucl. Acids Res. 22,
4009–15.
- Doss, R.P. (1996) Differential display without radioactivity – a modified
procedure. BioTechniques 21, 408–410.
- Luparello, C. et al. (1995) Parathyroid hormone-related peptide and 8701-BC
breast cancer cell growth and invasion in vitro. Evidence for growth-inhibiting and
invasion-promoting effect. Mol. Cell. Endocrinol. 111,
225–232.
- Luparello, C. et al. (1997) Clonal heterogeneity of the growth and invasive
response of a human breast carcinoma cell line to parathyroid hormone-related peptide
fragments. Carcinogenesis 18, 23–29.
- Luparello, C. et al. (2000) Use of M-MLV RT, RNase H-, Point Mutant, for
mRNA-differential display analysis of parathyroid hormone-related peptide (PTHrP)-treated
breast carcinoma cells.
http://www.promega.com/enotes
/applications/ap0019_tabs.htm
- pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual
#TM042, Promega Corporation.
(a)The PCR process is covered by patents issued and
applicable in certain countries. Promega does not encourage or support the unauthorized or
unlicensed use of the PCR process.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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Figure
1. Gel electrophoresis of five different recombinant plasmids (1–5) extracted
from JM109 lysates. |
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Figure
2. EcoR I restriction analysis of the recombinant plasmids shown in Figure
1. |
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