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Differential Display News |
| Volume 1, Number 1 |
December 1996 |
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Forum for Interaction on
Differential Display
Since the development of the differential display process in 1992, the application of this process has expanded into nearly all areas of science. One can find researchers using differential display to isolate novel genes in labs focusing on cancer research, embryogenesis, stress responses in plants, immune responses, studies of genetic diseases and a variety of other areas.
As the leader in reagent systems for differential display, GenHunter Corporation would like to provide an opportunity for regular interaction concerning this technique and its application. We recognize that when a developing technique is applied in such a variety of situations it is especially important to have a forum for the more basic as well as controversial issues to be discussed.
It is our hope that the publication of this newsletter will not only increase understanding of the technique itself, but also increase awareness of the breadth of possibilities for this technique and stimulate ideas for new projects. Literature reviews, technical questions, upcoming events, and relevant product releases are all on the agenda. We welcome ideas and contributions from our readers at any time. For instructions please see the contributions section on the bottom of page three.
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| Cold Spring Harbor Meeting a Success!
Many of our readers recently had the opportunity to participate in the second annual differential display conference held at Cold Spring Harbor Laboratory in Cold Spring Harbor, NY. More than 400 people were in attendance from 40 states and 25 countries. Not only was there diversity among the attendees themselves, but also in their projects.
The first half of the meeting was focused on methods of gene discovery. Several methods were discussed, focusing on differential display. Discussions revolved around reducing the number of false positives, enhancing reproducibility, cloning the correct product, and confirmation problems and solutions. Traditional differential display as well as modifications in PCR conditions, primer length, labeling procedures, and confirmation techniques were discussed.
The focus then shifted to the application of these powerful techniques. The speakers as well as those displaying posters were a testimony to the success of differential display and related methods as tools for gene discovery in a variety of laboratories around the world.
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Product Release
GenHunter is pleased to announce the new and innovative ReversePrime Kit (Cat. No. R701).
This kit provides a complete system for labeling cDNA for use in "Reverse Northern" blots
to conduct differential screening of positive clones generated by differential display.
It is ideal for either dot blot or colony hybridization screening of differential display
PCR products cloned into the pCR-TRAP® Cloning System (Zhang et al., Nucleic Acids Research,
1996, 24:2454-2455). This improvement significantly reduces the amount
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of labor required for screening by Northern Blot or Ribonuclease Protection Assay and provides high specificity.
It accurately identifies positives from false positives and reduces the amount of RNA needed
to conduct differential display screening. Cells are transferred via replica plating device
to membranes and processed to lyse the cells and allow the DNA to bind to the membrane.
The membrane is then probed with cDNA generated from the RNA samples and the autoradiograph
shows the location of colonies containing the insert of interest.
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Hundreds of articles have been published related to differential display.
It is not possible to comment on every one, but we have decided to highlight select, recently published
journal articles that would be of special interest to our readers.
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The development of the differential display process by Drs. Peng Liang and
Arthur Pardee in 1992 presented a breakthrough in the study of gene expression. However, as with
any new method, further refinement is necessary to streamline the process. Recent literature points
to the need for refinement in the area of dealing with a high rate of "false positives". The two
articles chosen to be reviewed here focus on improved methods to screen for true positive differences.
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Differential screening of gene expression difference
enriched by differential display. Zhang et al. Nucleic Acids Research, 1996.
24:2454-2455.
Zhang et al. (NAR 24:1996) have reported a breakthrough
in the quick and efficient identification of differentially expressed genes. In
the past, after performing differential display, researchers have been left with
the laborious task of screening a large number of clones before sequencing and
identification. The percent of false positive clones has been reported as high
as 90% and stems from several sources, such as co-migrating bands and inaccurate
band excision. The procedure outlined in Zhang et al. significantly reduces the
amount of labor and RNA required for screening.
The positive selection feature of the pCR-TRAP® Cloning System used by Zhang et
al. indicates that most colonies grown on a tetracycline plate following
transformation will have a PCR insert ligated into the vector. However, as
mentioned above, there may be more than one cDNA species in the PCR mix. To
identify which colonies contain the cDNA of interest, the colonies are transferred
onto two membranes by replica plating. The total RNA from the two cell types is
radioactively labeled by reverse transcription with an oligo-dT primer and used
to differentially screen the colonies on the membranes. The subsequent autoradiographs
indicate the location of colonies containing a
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differentially expressed insert
(appear only on the autoradiograph probed with RNA from one sample or the other)
and colonies containing a constituatively expressed insert (appear on both
autoradiographs). This procedure has been used to probe colonies generated from
bands expressed in an all or none fashion as well as bands which are expressed in
varying amounts. Many filters can be screened with the same probe, saving RNA,
time, and money.
Rapid selection and classi-fication of positive clones
generated by mRNA differential display. Vögeli-Lange et al. Nucleic Acids Research,
1996. 24:1385-1386.
Vögeli-Lange et al. present a new method for reverse
northern to allow fast and effective identification of positive differential display
clones. They have utilized the typical method of differential display including the
latest modifications in primer design and cloning strategy. Following cloning,
plasmids containing inserts of the correct size were amplified and slot blotted onto
duplicate membranes.The membranes were then probed with the corresponding original
DD-PCR reactions and the resulting hybridization pattern revealed differentially
expressed versus constituatively expressed cDNA fragments. To determine that the
differentially expressed fragments contain
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homologous sequences, one of the clones
was amplified with the original PCR primers, random prime labeled, and used to probe
all clones tested. The result showed high homology between the clones that had been
identified as differentially expressed and low homology with the remaining constituatively
expressed fragments.
This new method mimics the sensitivity of a PCR reaction. By using the original
(or freshly made with identical primers) PCR reactions as probes even clones of
differentially expressed rare messages can be detected as long as the PCR reaction
was able to detect this difference. Vögeli-Lange et al. compared this method
with a standard Northern hybridization and discovered that the standard Northern
failed to detect one of the six clones detected by the reverse Northern method.
This method does contribute to reducing the number of false positives generated
by differential display.
Although PCR is a reliable process, a possible drawback of this method is that there
are occasionally mispriming events that will present complications. Differentially
expressed bands that are created by PCR, when confirmed by the same PCR reaction,
will result in a false confirmation. Despite this possibility, this practical approach
to identifying positive clones may contribute to the ease with which differentially
expressed genes can be identified by way of differential display.
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Ask The Expert!
There has been a lot of discussion about the benefits of shorter
primers versus longer primers...what is the real issue? What length is optimal?
Contributed by: Peng Liang, Ph.D.
Vanderbilt Cancer Center
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The concept of differential display is to use a limited number of arbitrary primers
in combination with anchored oligo-dT primers to systematically amplify and visualize
most of the mRNA in a cell. The optimal length of arbitrary primers is determined by
the statistical consideration that each primer will recognize 50-100 mRNA species.
To do so, these primers must hybridize as 6-8mers. In practice, however, primers
shorter than 9 bases will not work for PCR amplification (Williams et al., 1991, NAR.
18:6531). Initially arbitrary decamers were used for differential display in combination
with two-base anchored primers. Although the method worked well in displaying the
expected number of mRNAs per primer combination, weak signals, fewer bands, and poorer
reproducibility were encountered by those who assembled the method under sub-optimal
conditions (likely due to variation in the quality of RNA samples, primers, MMLV,
isotopes, and Taq polymerase). These primers, as expected, hybridize initially to the
first strand cDNA as 6-8mers
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with most of the base-pairing occurring at the 3' end of
the primers.To determine the optimal length of arbitrary primers for differential display, the
following restrictions are essential: (1) the minimum primer length for PCR is 9
bases; (2) the arbitrary primers must hybridize in a predictable way in order to
rationally design a set of such primers to display most of the mRNAs in a cell; and
(3) the redundancy should be kept at a minimum. When taking into consideration the
above three restrictions, the optimal length for arbitrary primers becomes 13 bases
(Liang et al., 1994, NAR. 22:5763). Thirteen-mers are perfect PCR primers compared
to longer primers in regard to specific amplification of a target DNA sequence and
they offer more efficient priming than shorter primers. Since the first six to eight
3' bases of these 13mers provide the specificity of sequence recognition, they can be
rationally designed in such a way that all primers have maximally different 3' sequences
allowing a limited number to represent most of the mRNAs in a cell.
Some protocols claim that increasing the primer length to 20 bases or longer will
increase the "reproducibility" of the cDNA pattern displayed. One has to remember
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that longer primers are used to amplify specific genes, while shorter primers are used
to specifically amplify multiple genes. The longer primers under degenerate conditions
will hybridize in an unpredictable way, making the design of a representative set of
primers impossible. Although longer primers generate a complex cDNA pattern which may
appear reproducible, many of the bands may in fact represent the same mRNA due to the
"stickiness" of long primers when used under low stringency. In fact, A. Simpson and
colleagues have used this low stringency PCR for long primers to detect sequence
polymorphisms of the same gene (1994, PNAS, 91:1946). They first specifically
amplified the target genes under high stringency conditions, then, using the DNA
sequence specifically amplified as template and the same pair of long primers,
performed PCR under low stringency conditions. They were able to obtain a complex
pattern of PCR products from a single template thus indicating that the "more
reproducible nature" of long primers may be a trade off due to the redundancy of
the PCR products.
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 | Upcoming Events! |
| April 12-16, 1997 - American Association for Cancer Research annual meeting (San Diego, CA)
June 1997 - GenHunter is planning a workshop on differential display. Look for more information soon! |
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Notice: Contributions Welcome
To all users of differential display: The Differential Display News division of GenHunter Corporation encourages readers to contribute comments on relevant articles, particular technical insights, or new developments. Contributions selected for publication will receive a $200 credit for GenHunter reagents.
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Please send contributions to:
Differential Display News
GenHunter Corporation
264 Grassmere Park Drive, Suite 17
Nashville, TN 37211
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GenHunter Corporation
624 Grassmere Park Drive
Nashville, TN 37211
USA
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Phone: (800) 311-8260
Fax: (615) 832-9461
e-mail: genhunt@telalink.net
http://www.genhunter.com
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*The Differential Display process is covered by US patent 5,262,311 and other pending
patents licensed to GenHunter Corp.
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