Protocol:Restriction enzyme PCR

Principle
A small number of the &beta;-thalassaemia mutations create or abolish a restriction endonuclease recognition site in the globin gene sequence. Provided that the enzyme is commercially available (not always the case) and that there is not another site too close to the mutation, the loss or creation of a site can be used to diagnose the presence or absence of the mutation. This is useful for the diagnosis of a few of the common &beta;-thalassemia mutations, as listed in Table 5.6.

However the main use of this PCR technique is for the diagnosis of the clinically important Hb variants HbS, HbD-Punjab and HbO-Arab. Figure 5.9 shows the detection of the HbS mutation by this technique. The primer sequences used in the Oxford laboratory for diagnosing these Hb variants are listed in Table 5.7. However other primer sequences span the mutation site may be used. Whenever practicable the amplified product should include a second site for the appropriate restriction enzyme. This site will act as a control for the digestion reaction as it should be fully cleaved in product from both the normal and mutant DNA alleles. This is possible for the HbS and HbE mutations but not for HbO-Arab and HbD-Punjab, for which the flanking EcoR I sites are too far away from the one in codon 121

RE-PCR is also used for &beta;-globin gene haplotype analysis. At least 18 RFLPs have been characterised within the &beta;-globin gene cluster. However, most of these RFLP sites are non-randomly associated with each other and thus they combine to produce just a handful of haplotypes [29]. In particular they form a cluster that is 5' to the δ gene and a 3' cluster that extends downstream from the &beta;-globin gene. The DNA in between the two clusters contains a relative hotspot for meiotic recombination with a rate of approximately one in 350 meioses [30]. The &beta;-globin gene cluster haplotype normally consists of 5 RFLP's located in the 5' cluster (Hind II/&epsilon;-gene; Hind III/G&gamma;- gene; Hind III/A&gamma;-gene; Hind II/3’&psi;&beta;-gene; and Hind II/5'&psi;&beta;-gene) and two RFLPs in the 3' cluster (Ava II/&beta;-gene; BamH I/&beta;-gene) [31].

All of the seven RFLPs except BamH I site can be analysed very simply and quickly by PCR, using the procedure described in 3.2.3. The primer sequences and sizes of the fragments generated are listed in Table 5.8. The BamH I RFLP is located within a L1 repetitive element, creating amplification problems. A Hinf I RFLP located just 3' to the &beta;-globin gene is used instead, because these two RFLP’s have been found to exist in linkage disequilibrium [32].

Method

 * 1) Add the following to a 0.5 ml tube: 20 &mu;l PCR reaction mixture (as detailed in the ARMS protocol 3.2.1); 1 &mu;l each primer; 1 &mu;l genomic DNA (100 ng/&mu;l); 2 &mu;l sterile dH2O; 0.05 &mu;l of AmpliTaq DNA polymerase (5u/&mu;,l)
 * 2) Overlay with 25 &mu;l of mineral oil.
 * 3) Place in thermal cycler and perform 30 cycles of: 1 min at 94oC/1 min at 65oC/1.5 min at 72oC with a final period at 72oC for 3 min after the last cycle.
 * 4) Remove tubes and add 5-10 units of the appropriate restriction enzyme, plus 2 &mu;l of the corresponding 10x buffer.
 * 5) Incubate at 37oC for a minimum of 1 hour.
 * 6) Add blue dye, mix and spin as in protocol for ARMS-PCR.
 * 7) Load 20 &mu;l aliquot onto a 3% agarose gel consisting of 50% Nusieve GTC agarose and 50% ordinary agarose.
 * 8) Perform electrophoresis, stain and photograph as described for ARMS-PCR.

Table 5.6: &beta;-Thalassaemia mutations detectable by RE-PCR

Table 5.6: &alpha;-Thalassaemia mutations detectable by RE-PCR

Table 5.7 Oligonucleotide primers for the detection of &beta;S, &beta;E, &beta;D Punjab, and &beta;o Arab mutations as RFLPs

The annealing temperature for all primer pairs is 65oC Table 5.8