Differential display polymerase chain reaction

Differential display polymerase chain reaction (DD-PCR)1, uses PCR to amplify and display cDNAs derived from the mRNAs of a given cell or tissue type. First-strand-synthesis is primed with an anchored primer complementary to 13 nucleotides (nt) of the poly(A) tail of mRNA and the adjacent 2 nt of the transcribed sequence. Anchored primers therefore anneal to the junction between the poly(A) tail and the 3'-untranslated region of mRNA templates. A second primer, an arbitrary sequence of 10 nt, is then added to the reaction mixture, and double-stranded cDNAs are produced by PCR carried out at low stringency. By comparing the banding patterns of the resulting cDNA products, it is sometimes possible to identify the products of differentially expressed genes

Optimization of amplification conditions
1 | Set up a series of trial PCRs to establish the optimum concentrations of 'control' and 'test' RNAs required to produce a pattern of 100–300 amplified cDNA bands after gel electrophoresis and autoradiography. Make fivefold serial dilutions of the total RNA preparations in H₂O to produce concentrations between 1 g/ml and 100 g/ml.

2 | Choose one or more anchored 3' oligonucleotides and set up a series of annealing reactions that combine 8 l of template RNA (1–100 g/ml) with 2 l (6 pmol) of anchored 3' primer.

3 | Add the following to the annealing reactions:

Incubate at 37 °C for 1 h, then at 94 °C for 10 min to inactivate the reverse transcriptase.

Control for contaminating DNA by setting up one or more reactions without reverse transcriptase and carry through Step 7. If necessary, treat the RNA with RNase-free DNase I.

4 | Set up two series of eight 0.5-ml amplification reactions. Each tube should contain the following:

To each tube, add 2 l of a different arbitrary 5' primer. Mix by tapping the sides of the tubes.

5 | Into one series of eight tubes, dispense 3-l aliquots of the reverse transcriptase reaction containing the test RNA. Into the other series of eight tubes dispense 3-l aliquots of the reverse transcriptase reaction containing the control RNA. Mix the contents and place in the thermal cycler.

6 | Amplify the nucleic acids using the following program:

Times and temperatures may need to be adapted to suit the particular reaction conditions.

7 | Separate the radiolabeled products by electrophoresis through an electrolyte gradient polyacrylamide gel of the type used for DNA sequencing. Continue electrophoresis at constant electrical power until the tracking dye has migrated about two thirds of the length of the gel. Dry the gel and expose it to autoradiographic film.

Examine the pattern of DNA bands; a good display contains between 100 and 250 well-resolved bands. Select the conditions that work well with the largest number of primer pairs.

Amplification of cDNAs for differential display
8 | Repeat the annealing, reverse transcription and amplification reactions using all combinations of primer pairs and the optimum amount of RNA templates and separate products as in Step 7. Compare the bands obtained with each primer pair from the different RNA populations and identify differentially expressed bands.

9 | Using the autoradiograph as a guide, cut target bands from the dried gel. Soak each gel sliver overnight in a separate 0.5-ml tube containing 50 l of sterile H₂O.

10 | Puncture the base of each tube with a small-gauge needle and place each tube inside a 1.5-ml microcentrifuge tube. Centrifuge for 20 s to transfer the eluate into the larger tube.

11 | Amplify the eluted fragment in a reaction containing the following:

12 | Place the tubes in a thermal cycler and amplify the nucleic acids using the program from Step 6. Polymerization should be carried out for 1 min for every 1,000 base pairs of length of the target DNA.

13 | Estimate the concentration of the reamplified DNA fragment by analyzing 5–10% of the product on a 1% agarose gel.

14 | Amplified DNA products can now be ligated into a dT-tailed vector for transformation into Escherichia coli and further analysis.

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Source
This protocol was adapted from Differential Display-PCR in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) 8.96–8.016 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001; http://www.cshlpress.com/link/molclon3.htm). This protocol was provided by Charles P. Landrum (University of Texas Southwestern Medical Center, Dallas, USA).
REFERENCES
Liang, P. & Pardee, A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971 (1992). | PubMed | ISI | ChemPort |
Liang, P. & Pardee, A.B. Differential display. A general protocol. Methods Mol. Biol. 85, 3–11 (1997). | PubMed | ChemPort |