Protocol:Gap-PCR

Principle
Gene deletion mutations in the &beta;-globin gene cluster may be detected by PCR using two primers complementary to the sense and antisense strand in the DNA regions that flank the deletion. For small deletions of less than approximately 1 kb, the primer pair will generate two products, the smaller fragment arising from the deletion allele. For large deletions, the distance between the two flanking primers is too great to amplify the normal allele and product is only obtained from the deletion allele. In these cases the normal allele is detected by amplifying across one of the breakpoints, using a primer complimentary to the deleted sequence and one complimentary to the flanking DNA.

Gap-PCR is used to diagnose some &delta;&beta;-thlassaemia deletions, HPFH deletions, &alpha;-thalassaemia deletions (Table 1) and also the triple &alpha;-gene locus generated by the 3.7-kb single &alpha;-gene deletion [5]. A typical gap-PCR test is illustrated in Figures 1 and 2. For the diagnosis of &alpha;-thalassaemia, the primers can now be multiplexed. The 3.7-kb and 4.2-kb &alpha;+-thalassaemia deletions can be detected in one assay (Table 2), the --MED and -(&alpha;)20.5 &alpha;o-thalassaemia deletions in one assay (table 3) and the 3 Southeast Asian &alpha;o-thalassaemia deletions in one assay (Table 4). The protocols used in the Oxford laboratory for the multiplexing of these primers are given in Tables 2-4, but it should be noted that the quantity of each primer pair relative to the others may need adjustment to gain optimum amplification of all the products. PCR diagnosis of the triple &alpha;-gene (anti 3.7 allele) requires two separate assays (Tables 5 & 6). The presence of the allele is diagnosed from a comparison of the results of each assay run side-by-side and the genotype of the DNA sample can be deduced (Table 7) in most instances.

Table 1 Thalassaemia deletion mutations which have been diagnosed by gap-PCR

Method

 * 1) Set up the reaction mixture to a final volume of 22 &mu;l into a 0.5 ml tube with the following components as required: 1 &mu;l genomic DNA (100 ng/&mu;l) (1 &mu;l of forward primer - flanking sequence (10 pmol/&mu;l), 1 &mu;l reverse primer - flanking sequence (10 pmol/&mu;l),1 &mu;l of primer - deleted sequence (10 pmol/&mu;l), 1 &mu;l of primer - inverted sequence (10 pmol/&mu;l), 2.5 &mu;l of 1.25 mM (dNTP mixture), 2.3 &mu;l of 10x Gap PCR buffer as recommended for the primers in the original reference (see Table 5.2) and below.
 * 2) The buffer recommended for the &alpha;-thalassaemia primers is 750 mM Tris-HCl pH 8.8, 200 mM (NH4)2 SO4, 0.1% Tween 20). The buffer for the &alpha;-thalassaemia primers should also contains 0.5 M betaine and 0.5% DMSO (this can be achieved by adding 2.5 &mu;l of 5 M betaine and 1.25 &mu;l 10% DMSO).
 * 3) Make up all reactions to a final volume of 22 &mu;l by adding sterile dH2O.
 * 4) Overlay with 25 &mu;l of mineral oil.
 * 5) Prepare enzyme mixture: 0.2 &mu;l reaction buffer (10x), 0.1 &mu;l AmpliTaq (5U/&mu;l) (PE Biosystems) for the &beta;-gene primers, 0.1 &mu;l Platinum Taq (5U/&mu;l) (Invitrogen) for the &alpha;-gene primers, and 2.7 &mu;l sterile dH2O, to make 3 &mu;l.
 * 6) Mix enzyme mixture and hold on ice.
 * 7) Place reaction mixtures in thermal cycler and perform one cycle as follows, adding 3 &mu;l of the enzyme mix after 2 minutes of the 94 oC denaturation step: 4 min at 94oC/1 min at 55-65oC(as recommended)/1.5 min at 72oC
 * 8) Continue for 33 cycles with the following steps per cycle: 1 min at 94oC/1 min at 55-65 oC (as recommended in the published references or in tables 5.9-5.13)/1.5 min at 72 oC
 * 9) Finish with one cycle as follow: 1 min at 94 oC/1 min at 55-65oC (as recommended)/10 min at 72oC.
 * 10) Hold at 15oC until gel electrophoresis.
 * 11) Remove tubes from thermal cycler. Add 5 &mu;l of blue dye, mix and centrifuge.
 * 12) Depending on expected product sizes, load a 20 &mu;l aliquot onto a 1-3% agarose gel and run at 100 V for 45 min to 2 hrs in 1x Tris-borate-EDTA buffer (TBE).
 * 13) Stain gel in ethidium bromide solution (0.5 &mu;g/ml) for 15-30 minutes, visualise bands on a UV light box (312 nm) and photograph with an electronic camera system or a Polaroid CU-5 camera fitted with an orange filter (e.g. Wratten 22A). For guidance re interpretation see notes 6,7 and 8.

Materials

 * 1) DNTPs: Add together 50 &mu;l of a 100 mM solution of each dNTP (as purchased) and 3.8 ml of distilled water. The 1.25 mM dNTP stock solution should be stored in frozen aliquots.
 * 2) 10x Gap PCR reaction buffer (composition varies according to primers used) see methods and Table 2
 * 3) Betaine (Sigma-Aldrich Chemical Co Ltd, England)
 * 4) Mineral Oil to overlay PCR reactions
 * 5) PCR primers: dilute aliquots of primer stock solutions to make a working solution of 1 OD unit/ml and store frozen.
 * 6) Ammonium sulphate buffer: 75 mM Tris-HCl (pH 9.0), 20 mM (NH4)2SO4, 2.0 mM MgCl2, 0.01% Tween 20, 10% DMSO, 10 mM &beta;-mercaptoethanol (all final concentrations).
 * 7) Taq polymerases and 10x Taq buffers: in my laboratory are as follows, AmpliTaq Gold (PE Biosystems) works best for ARMS-PCR/RE digestion assays and Platinum Taq (Gibco Life Technologies) for gap-PCR.
 * 8) Tris-borate -EDTA (TBE) buffer : 89 mM Tris-borate, 89 mM boric acid, 10 mM EDTA, pH 8.0.
 * 9) Blue running dye (15% ficoll/0.05% bromophenol blue).
 * 10) UV transilluminator and Polaroid camera, or UV electronic camera system
 * 11) 0.5 &mu;g/&mu;l Ethidium bromide

Multiplex Gap-PCR
Specific primer details etc are listed below for the multiplex diagnosis of the common &alpha;-thalassaemia genotypes and the triplicated &alpha;-globin allele. Figures 5.10 and 5.11 show example results all the common &alpha;-thalassaemia geneotypes.

Multiplex PCR protocol for the diagnosis of -&alpha;3.7 and -&alpha;4.2 deletions
      Run PCR products out on 1.5% (1:1 Nusieve:agarose ) gel for 2-3 hours    
 * Primer sequences [1]
 * PCR reaction mix
 * Gel electrophoresis conditions
 * Interpretation of results

Multiplex PCR protocol for the diagnosis of the --MEDand -(&alpha;)20.5deletions
      Run PCR products out on 2% (1:1 Nusieve:agarose ) gel for 1-1.5 hours </li>  </li> </ol>
 * Primer sequences [2]
 * PCR reaction mix
 * Gel electrophoresis conditions
 * Interpretation of results

Multiplex PCR protocol for the diagnosis of the -- SEA / --FIL / --THAI &alpha;o-thalassaemia deletions
  </li>  </li>  Run PCR products out on 1.5% (1:1 Nusieve:agarose ) gel for 2 hours </li>  </li> </ol>
 * Primer sequences [3]
 * PCR reaction mix
 * Gel electrophoresis conditions
 * Interpretation of results

Reaction Mix 1
  </li>  </li>  Run PCR products of reaction mixture 1 out on 2% (1:1 Nusieve:agarose ) gel for 2 hours, in lane next to those of reaction mixture 2. See Table 5.14 for interpretation of results. </li>  </li> </ol>
 * Primer sequences
 * PCR reaction mix
 * Gel electrophoresis conditions
 * Product sizes

Reaction Mix 2
  </li>  </li>  Run PCR products of reaction mixture 2 out on 2% (1:1 Nusieve:agarose ) gel for 2 hours, in lane next to those of reaction mixture 1. See Table 5.14 for interpretation of results. </li>  </li> </ol>
 * Primer sequences
 * PCR reaction mix
 * Gel electrophoresis conditions
 * Product sizes

Interpretation of the results of reaction mixes 1 & 2 for the diagnosis of the &alpha;&alpha;&alpha; (anti 3.7) allele
<ol> <li>
 * Products (bp)

Notes: </li> <li> </li> </ol>
 * 1) &alpha;&alpha; allele: amplifies with C3+C10 (1.9kb) and C2+C10 (2.1kb).
 * 2) The -&alpha;3.7 allele: amplifies with only C2+C10. Gives a shorter product (1.9kb) than normal because of the deleted &alpha;-gene.
 * 3) The &alpha;&alpha;&alpha; allele: amplifies with C3+C10 (1.9kb) and twice with C2+C10 (2.1kb) because of the extra &alpha;-gene.
 * Possible gel patterns

Figure 1. The diagnosis of &alpha;+-thalassaemia deletion mutations by multiplex GAP PCR using the primers described under Primer sequences [1] above.



Figure 2 The diagnosis of &alpha;o-thalassaemia deletion mutations by multiplex GAP PCR using the primers described under Primer sequences [2] & [3] above.