• Purpose
• Method
• What makes it a great technique?
• Overview

        DisplayPROFILE^TM RFDD is a method which uses the polymerase chain reaction (PCR) to make consistent and rapid gene expression profiles and to identify differentially expressed genes in various tissues and cells. To produce molecular snapshots of expressed genes, the displayPROFILE^TM technique involves digesting double-stranded cDNA with a restriction enzyme, ligating specific DNA adaptors to the cDNA fragments, and annealating specific primers onto the adaptors (Display Sytems Biotech).  The cDNA fragments are then divided into 64 different PCR reactions. Each PCR reaction amplifies a different expression window, or a set of 400 or more cDNA fragments.  Unlike standard differential display systems that use poly-A primed PCR amplification, RFDD-PCR uses the TaqI restriction enzyme and specific PCR primers for amplification (Display Systems Biotech). As a result of not relying on poly-A-primed PCR, this technique can be used for both eukaryotic and prokaryotic systems. The displayPROFILE^TM technique is important in the biotechnology world, for PCR methods can be used in the diagnosis of infectious diseases. In particular, this technique could possibly be used for cancer research since specific genes that are expressed or repressed in diseased versus healthy tissues can be easily identified and compared. Using RFDD-PCR, the genetic basis of specific cancers and new cancer therapies could be discovered.

How is it performed?

  The displayPROFILE^TM RFDD technique involves five major steps:

            1.   Double -stranded cDNA is synthesized from specific mRNA sequences

            2.   Double-stranded cDNA is digested with TaqI restriction enzyme. Digestion with TaqI produces
                  sticky ends on the cDNA fragments.

            3.   Two specific DNA adaptors are ligated onto the sticky ends of the cDNA fragments.
                  One of the adaptors is called the EP-adaptor, which consists of a 5'-overhang and an
                 "extension-protection group" on its 3'-end. The purpose of the EP group is to ensure that the
                  5'-overhang  will not be filled in by 3' to 5' synthesis and that cDNA fragments with EP adaptors at both
                  of its sticky ends will not be amplified during the polymerase chain reaction. The second adaptor used
                  is called the standard adaptor. Only those fragments that have the EP adaptor ligated onto one
                  of its ends and the standard adaptor ligated onto its other end are amplified using PCR (Display
                  Systems Biotech).

            4.    Sixty-four high-strigency PCR reactions are carried out with  0-extension 5'- primers and 64 different
                   displayPROBE 3'-primers. A  0-extension 5'-primer is annealed to the EP adaptor on the
                   cDNA fragment, while one of the displayPROBE primers is annealed to the junction between the cDNA
                   insert and the standard adaptor.  Because a displayPROBE primer consists of three bases
                   that extend into the insert cDNA, amplification of cDNA fragments with a standard adaptor
                   ligated onto both ends is prevented (both ends would need the same three bases to be amplified), and
                   each displayPROBE is specific for a certain cDNA sequence. Thus, in order to amplify all fragment
                   variations, sixty-four displayPROBE primers are needed for each PCR reaction (4^3=64) (Display
                   Systems Biotech).
                   When PCR is run, each of the 64 reactions amplifies an "expression window," which
                   consists of 400 or more fragments. Because each cDNA fragment can be oriented in two different
                   orientations relative to the two adaptors, each amplified cDNA fragment should be represented twice-
                   once in two different expression windows.  See figure below:
 
 

            5.    The amplified gene fragments are separated on a gel (Display Systems Biotech).
 
 

  OVERVIEW:
 
 

Why is displayPROFILE^TMsuch a great technique?
 

• First,  RFDD-PCR is a breakthrough technique, for it allows the identification of differentially expressed genes in both eukaryotic and prokaryotic systems. This is because RFDD-PCR  relies on restriction enzyme digestion and specific PCR primers for amplification rather than poly-A-primed PCR amplification. The only difference between the two systems is that only 32 expression windows are created with prokaryotic systems while 64 expression windos are necessary for eukaryotic systems as a result of prokaryotes having smaller amounts of mRNA (Display Sysytems Biotech).
• RFDD-PCR generates cDNA fragments using the TaqI restriction enzyme, which ensures that only coding regions are amplified when performing PCR (Display Systems Biotech).  This is an advancement in PCR expression profiling techniques, for standard differential display systems often amplify 3'-untranslated regions of genes as a result of relying on poly-A-primed PCR.
• As a result of using primers that have known sequences, the PCR profiling information can be used to predict in which expression window your gene of interest will appear. The identity of your gene of interest can then be concluded.
• The identity of the differentially expressed genes can be confirmed because each amplified fragment appears twice in a profile (Display Systems Biotech). The identification of each fragment at two positions in a profile serves as an internal control. Because of this internal control, your results are highly reliable.
• Stringent PCR conditions produce highly resolved bands (Display Systems Biotech).
• The RFDD-PCR method is a great biomedical research technique, for the changes in gene expression profiles as a disease progresses can be analyzed using the generated molecular snapshots.

Carrie's Homepage

References:

Brown C. 1995. Homepage. <http://falcon.cc.ukans.edu/~jbrown/pcr.html>. Accessed 2000 Feb 20.

Display Systems Biotech. Homepage. <http://www.displaysystems.com>. Accessed 2000 Feb 20.

Display Systems Biotech. DisplayPROFILE^TM-Expression profiling using Restriction Fragment Differential Display (Brochure).

Macelis D, Roberts RJ. 2000 Feb 17. TaqI TypeII restriction enzyme. <http://rebase.neb.com/rebase/enz/TaqI.html>.
Accessed 2000 Feb 20.

Please send comments to: casmith@davidson.edu