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Over 75 primer pairs were chosen and a number of multiplex mixtures were designed and used for different purposes. Examples of all multiplex mixes are presented below.
(All unlabeled gel lanes are the marker: 1 kb DNA ladder (GIBCO). At the bottom of each image, the PCR buffer concentration used is also indicated)
Fig. 1. Mixtures A-E on an a 2.5% agarose gel. Arrow indicates the presence of an unspecific product. Although not desirable, this product did not interfere with the use of mix E in a microdeletion screening project. Occasionally, mix C was used without the primers for loci 4 and 5. In such cases, this mix is called mix C*.
Fig. 2. Mixtures F-I on a 2.5% agarose gel.
Fig. 3. Multiplex PCR with mix J on four different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. Primers amplify nonpolymorphic loci. The sizes of the longest and the shortest product are also indicated.
Fig. 4. Multiplex PCR with mix K on eight different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. These primers amplify polymorphic loci, and alleles of different sizes can be observed. The shortest alleles of locus 6 and the longest alleles of locus 7 can, sometimes, overlap, making it difficult to assign precisely their origin. The sizes of the longest and the shortest product are also indicated.