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Comparison of agarose type (non-polymorphic loci)
Two types of agarose from the same manufacturer (both in use in this laboratory) were compared for their efficiency in separating the multiplex PCR products (Fig. 38). Multiplex PCR with primer mixtures A (one sample) and F (4 samples) was performed. Same amount of each reaction was loaded on a 3% agarose gel of each type. Electrophoresis time was about 1.6-1.7x longer for the regular (SeaKem LE) agarose gel.
In accordance to the manufacturer's specifications, the NuSieve agarose separates short products better than the regular agarose, and in a reduced amount of time. Although the gels had the same thickness, results also indicate that the "special" NuSieve agarose is more transparent than the regular agarose. Although NuSieve agarose is much more expensive, it provides some cost reduction by requiring less amount of agarose for the same separation power and by requiring less amount of separation time. These particular advantages can make such "specialized" agaroses useful for particular applications.
It is worth mentioning that other agaroses (from different manufacturers) used, perform similarly.
Fig. 38.Separation of the same multiplex products of mixtures A and C (four lanes) on two different agaroses. Arrow indicates a few unspecific products in lane 2 and circle indicates primers (or primer-dimers), both of these being stronger on the NuSieve gel. This shows tthat NuSieve gels have a higher transparency. Also, separation on NuSieve gels was achieved in les amount of time, over a shorter gel length. The unmarked lane(s) is the 1 kb ladder (GIBCO).
Agarose gel (running time)
Agarose gels can be run at various voltages, depending on the separation desired and the available time. It was noticed that, at least for PCR products smaller than 600 bp, separation is better and bands are sharper if gels are run very fast (3-4 hours for a 15-20 cm long 2-3% agarose gel). When the same gel runs at a low voltage overnight (14-16 hours) the products become less separable or "puffy" due to the diffusion in the gel (compare Fig. 39 below with lane C in Fig. 1).
Fig. 39.Multiplex PCR with mix C was performed on 9 DNA samples to screen for microdeletions (chromosome Y loci. Gel separation was performed overnight (14 hours). Products appear diffuse, less intense, and less separable (product 1 and 2 are "fused" together). Green and magenta arrows indicate lack of loci #1 and # 4 (microdeletions) in some of the DNA samples tested. he unmarked lane(s) is the 1 kb ladder (GIBCO).
Agarose gels and polymorphic loci
As depicted also in Fig. 7, agarose gels can be used to separate PCR products of plymorphic loci. In most cases, two or three bands appear, due to heteroduplex formation between the long and short alleles. However, separation of multiplex PCR reaction products of many polymorphic loci (for example mixture K) coud become a problem for nondenaturing agarose gels. In mixture K, products were chosen so they differ by no less than 5 bp and no more than 45 bp. As depicted in Fig. 7 and Fig. 40 (below), agarose gels do not have sufficient separation power. Bands become to overlap and it is difficult or impossible to find and label each band. Denaturing polyacrylamide gels are recommended in such cases (see below).
Fig. 40.Multiplex PCR amplification of mixture K, using four different DNA samples. A 2.5% agarose gel was used to separate the products. As depicted also in Fig. 7, 2-3 bands become visible for each product. When together, many of these bands start overlapping, making identification of individual products/alleles impossible. The unmarked lane(s) is the 1 kb ladder (GIBCO).
To separate PCR products differing in only a few bp in length (for example, microsatellite markers), 6-10% PAA gels need to be used. Whereas non-denaturing PAA gels work very well for non-polymorphic loci, unusual bands appear when microsatellites are separated on this type of gels. For example, in an analysis of two polymorphic loci from two hybridomas, each carrying one copy of a human chromosome, for each locus tested there were 2 bands on the non-denaturing PAA gel (Fig. 41). It is unclear where the extra band originates when only one allele is amplified.
Fig. 41.Multiplex amplification of two loci on DNA from two human-rodent cell lines, each with a different copy of a human chromosome, and their combination (a+b). Although in lanes a and b each locus yields only one allele (i.e. one band), on a non-denaturing polyacrylamide gel each of the two expected products (arrows) was acompanied by another one running slower on the gel (oblique arrows). A similar aspect persisted in lane A+B. Lanes 1 and 2 show separation of products of mixture K on two different genomic template DNAs. The unmarked lane(s) is the 1 kb ladder (GIBCO).
Denaturing PAA gels
Denaturing 6% PAA/7M urea sequencing gel can be easily used to separate radiolabeled multiplex PCR products, whether these are polymorphic or unique. Denaturing PAA gels, however, are more expensive, time consuming and might prove technically more difficult. Figure 3 below, shows separation of the unique products of multiplex mixture J; double bands are visible for some of the loci. Figure 4 below shows separation of the polymorphic loci (microsatellites) of multiplex mixture K; two distinct alleles are visible for many of loci in the 8 DNA samples tested.
Fig. 3 (duplicate). 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 (duplicate). 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.