Because the oligonucleotides that are used are degenerate, there are no "standard" reaction conditions that will invariably work. The reaction parameters
Atr II TTEHDIYKLPRMRHPNILEFLGVEKHMDKPEY--WLISTYQHNGSLCDYLKSHTISW
ActR-IIB2 QSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITW ActR-II QNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANWSW
Atr II PELCRIAESMANGLAHLHEEIPASKTDGLKPSIAHRDFKSKNVLLKSDLTACIADFG
ActR-1IB2 NELCHVAETMSRGLSYTHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFG ActR-II NELCHIAETMARGLAYLHEDIPGLK-DGHKPAISHRDIKSKNVLLKNNLTACIADFG
Atr II RCDFA-GPVGEFQLPFEAELGLRPSLDEVQESWMKKLRPRLLNSWRAHPGLNVFCD
ActR- IIB2 RCKAADGPVDEYMLPFEEEIGQHPSLEELQEVWHKKMRPTIKDHWLKHPGLAQLCV ActR-11 RCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVWHKKKRPVLRDYWQKHAGMAMLCE
Fig. 2. (A) portion of the amino acid sequences of the Drosophila Act-R gene and the mouse Type II Activin receptors (3). The motifs shown in bold are those on which the PCR primers are based.
RT-PCR on Embryos B
5' external primer -1 -5' CGGAATTCAGATTCGGATGCGTATGG 3 '
3' external primer - 2-5' GCTCTAGACTCAGCATCATGATCCCA 3 '
internal primer - 3 -5' GCTCTAGAAAAAACCTCAGGAGCCAT 3 ' C GC T C C
Fig. 2. (continued) (B) The sequences of the primers are shown underneath. Note that the 5'-primer also has an added £coRI site and the two 3'-primers an Xbal site.
must be empirically determined for each primer pair. The two variables that are most frequently altered are the concentration of MgCl2 in the reaction and the temperature at which the oligonucleotides anneal to the substrate. In a typical reaction, the author would initially try three final concentrations of MgCl2, which span the normal range that is usually effective—1, 1.5, and 2 mM. If these concentrations are not effective, then concentrations from 0.5-5 mM can also be tested. In the case of exact match primers, one tends to use an annealing temperature that is 10°C below the melting temperature of the oligo. This is obviously not applicable to degenerate oligos, which are a mix of oligos with a range of melting temperatures. Ideally, the higher the annealing temperature that can be used, the greater the specificity of the reaction but the temperature should also be low enough to allow the oligos to anneal to all of the potential target sites of interest. A useful annealing temperature to start with is 50°C. If at this temperature no sequence is amplified then the temperature can be dropped even to as low as 37°C, although reactions using these lower temperatures will invariably produce spurious amplification products at a much higher frequency. If annealing at 50°C produces multiple amplified products, then the annealing temperature should be raised. Some of the background may be
owing to the primers annealing and Taq extending at lower temperatures as the reaction mix warms up. One way to reduce this sort of background amplification is to "hot start" the reaction. This is achieved by adding the Taq poly-merase to reaction mix that has been denatured at 94°C and then cooled to 55 °C or less effectively, but more simply by making up the complete PCR reaction mix, including Taq, on ice and then transferring the tubes straight to a block that has been prewarmed to 94°C.
The PCR reaction consists of the following components: Taq polymerase buffer, MgCl2, deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) at a final concentration of 0.2 mM, oligonucleotide primers that should be in excess and are often used at concentrations of 0.5-1 mM, 1 U of Taq poly-merase, target cDNA, and H2O to make up the volume.
A typical PCR reaction of 100 |L using Taq polymerase purchased from Promega would be as follows:
1. Add the following to a microcentrifuge tube:
10X Reaction buffer (Mg2+ free) 10 |L.
25 mM MgCl2 4 |L (1 mM), or 6 |L (1.5 mM), or 8 |L (2 mM).
External primers (at a concentration of 0.5 |g/|L approx 50 |M) 1 |L of each.
2. Overlay with 50 |L of paraffin oil to prevent evaporation. This step is not necessary if one is using a thermal cycler with a heated lid.
3. Place the sample in the thermal cycler and carry out the amplification. A typical amplification protocol is:
Subsequent cycles (x30)—denature, 94°C for 30 s.
Final cycle (x1)-extend 72°C for 10 min.
4. For the second round of amplification, use the internal primer and the appropriate external primer, and use 5 | L of the completed first-round PCR reaction mix as the DNA substrate. Mix the reagents and carry out the amplification as for the first round again with the range of Mg concentrations. One other factor that can be varied is the amount of first-round reaction that is added as a substrate for the second round. In some instance, the second round may be a lot cleaner if considerably < 5 |L is added.
5. At the end of the second round, run a 10-|L aliquot of all samples, i.e., all Mg concentrations from both the first and second rounds, on an agarose gel. Figure 3 shows the results obtained with the activin type II receptor primers. As can be seen the first round of amplification with the external primers did not produce a band at any of the Mg concentrations used, but in the second round with the internal primer and the 5'-external primer, a fragment of the predicted size was
Fig. 3. Ethidium bromide-stained agaraose gel electrophoresis of the products PCR amplification. M is the marker track (1-kb ladder from Gibco-BRL), 1-3 are from the first round of amplification and 4-6 are from the second. The reactions run in lanes 1 and 4 were at 1 mM MgCl2, 2 and 5 at 1.5 mM, and 3 and 6 at 1.5 mM.
amplified under all Mg concentrations, although the 1-mM concentration was clearly more efficient.
If the amplification has worked and an appropriate-sized fragment is present, the PCR reaction mix should then be cleaned up by phenol/chloroform extraction, especially if using oil, then ethanol-precipitated and redissolved in a small volume, and then digested with the appropriate restriction enzymes. The digest should then be run on a gel, the fragment isolated and subcloned into an appropriate vector, and the products sequenced. In some cases, all of the resulting clones will contain fragments of members of the gene family of interest, but in a number of cases, one will also find that other unrelated products have been amplified. This last point merely serves to stress the importance of sequencing the products of the amplification.
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