differential display
5.1 A modified differential display protocol
As has already been discussed, one of the reasons for the high background of non - differential clones observed in conventional differential display experiments could be that the primer used for reverse transcription is also used in the PCR at a stringency where every species that is reverse transcribed can be amplified, if only
asymmetrically. Consequently at any position in the gel, there could exist a large population of species that may or may not be visible but which could be reamplified. This latter possibility is further enhanced by the reamplification conditions also being at low stringency. Even if reamplification is carried out with 5' extended primers, the first two cycles are at low stringency and so, in effect, conditions similar to
differential display exist in this reaction. An obvious remedy for this problem would be to somehow remove the reverse transcription primer from the subsequent PCR. When dealing with very small amounts of RNA/cDNA, it is not desirable to attempt this using physical methods because of the risk of losing material. The same effect could be achieved biochemically if the reverse transcription primer could be effectively barred from participation in the PCR on account of a high annealing stringency after the first two cycles.
The principle of this idea is shown in figure 4.1a. If reverse transcription is carried out with a conventional dT24 primer and the subsequent PCR performed with a single
arbitrarily chosen 24mer such as those used earlier for remplification, then after two low stringency PCR cycles that allow the primers to be incorporated into second
Fi^ r e 4.1
(a) Schematic representation of a modified RNA fingerprinting protocol designed to prevent the primer used in the reverse transcription step from taking part in the PCR. After reverse transcription with a dX24 primer, a single arbitrary 24mer is used for
PCR with 2 cycles of low stringency followed by 40 cycles at high stringency. This latter step is performed at an annealing temperature at which the dT24 primer will not
bind the template and initiate DNA synthesis, although it may take part in the first two cycles of PCR. The fingerprint observed is derived from the amplification of those cDNAs generated by the 24mer after two cycles of low stringency annealing. If the reverse transcription primer is effectively removed from the PCR then the
background of cDNAs which are not differential may be diminished.
(b) Autoradiograph of differential display fingerprints of 9.5 dpc embryo RNA using the dT24 primer for reverse transcription and PCR with the protocol outlined in figure
4.1a. The reaction products were separated by electrophoresis through a 5% (w/v) native polyacrylamide gel as before. The tracks represent the products seen when the high stringency annealing cycles are carried out at 45°C, 50°C, 55°C and 60°C respectively. As the annealing temperature rises above 50“C, reaction products disappear indicating that the dT24 primer does not participate efficiently in the
reaction.
(a)
mRNA A A A A A A A A A A A A n r n ' T V T i r fv t;;;. 5’ Reverse transcription F T T T T T T T T - 5 ’ 5’ga" r; Low stringency PCR 2 cycles High stringency PCR 40 cycles ÜTAÜTTCGT': c;: t a g g a c t g ît a g s’ §mm Polyacrylamide gel electrophoresis RT PCR lin gerp h n t(b)
Size (Bp)
Reverse Transcription and PCR with dT2 4
Annealing temperature (°C) 45 505560
341
258
strand cDNA, the annealing stringency can be raised so that only PCR products with the arbitrary primers at their ends are amplified and those with the reverse
transcription primer can be excluded. This should remove the problems of multiple clones arising from single bands and of clonable species being amplified from apparently blank areas of the differential display gel if these problems are indeed due to the participation of the reverse transcription primer in the PCR step. The idea of using longer primers in the differential display has been postulated before (Ayala et al, 1995; Diatchenko et a l, 1996a), though it does not appear to have been widely adopted. AP-PCR approaches use long arbitrary primers to generate differential PCR amplicons, but they involve reverse transcription with one of the primers used, and so the same theoretical problems arise. The use of longer primers in the display PCR should also lead to larger reaction products (because there should be fewer sites in a given cDNA where an arbitrary 24mer can anneal), and the reamplification step can be carried out at high stringency, thereby eliminating internal priming. In the protocol proposed here, the RNA population is not subdivided at the time of reverse
transcription with anchored primers. This may not be necessary as a reduced number of bands per reaction are predicted by the use of longer primers (Diatchenko et al.,
1996a).
In order to ascertain whether it is possible to eliminate bands solely due to the reverse transcription primer from the resulting cDNA fingerprint, 50ng 9.5 dpc mouse
embryo RNA was reverse transcribed with a dT24 primer and the products PCR
amplified with the dT24 as the only primer. The reaction conditions included two
cycles of low stringency annealing (40°C) followed by 40 cycles of annealing at 45°C, 50°C, 55°C or 60°C. The reaction products were resolved by electrophoresis and an autoradiograph is shown in figure 4.1b. It can be easily appreciated that visible bands disappear at higher temperatures, suggesting that at 60°C the reverse
transcription primer has reduced participation in the PCR reaction.