Dealing With Very ATRich and Very GCRich Sequences

Genomic DNA is diverse in its GC-content and it is hard to imagine that the same PCR conditions working perfectly for any given genomic locus. In fact,

Fig. 2. Genotyping of 20 DNA samples (4 ng each) and 4 NTCs with rs454557 (Table 1, no. 7). Reaction volumes were 10 ||L.

we have observed that our standard conditions work reasonably well for approx 80% of SNPs in human genome. About 20% of SNPs, however, are located in loci that are either too AT-rich or too GC-rich. To estimate %GC of the sequence, calculate the number of Gs and Cs in the area from 50 bases 5' of the polymorphism to 50 bases 3' of it. If a sequence has 35 or less GCs, it is an AT-rich sequence. If the sequence has 65 or more GCs, it is a GC-rich sequence.

Very low signal may be an indication that you are dealing with an AT-rich sequence. Try to increase Mg2+ concentration to 2.5-3.0 mM by adding approx 25 or 50 mM MgCl2 solution to the amplification cocktail. Examples of SNP assays that required 2.5-3.0 mM Mg2+ are presented in Table 5.

Strong signal in all reactions including the NTCs may be an indication that you are dealing with a GC-rich sequence. Try adding betaine to 0.25 or 0.5 M concentration. When running the assays for GC-rich sequences in 0.5 M betaine, decrease the annealing temperature in Amplification-step 2 to 45°C. If adding betaine does not help, you may need shorter primers (see Subheading 3.1.1.4., step 4). Examples of SNP assays that required betaine due to high GC-content are shown in Table 6.

It should be noted, however, that prediction of the outcome solely from the %GC is not always straightforward and some notable exceptions were found. Thus, rs5498 (60% GC) and rs887586 (62% GC) are by no means AT-rich sequences; nevertheless, these SNP assays worked well only under conditions suited for the AT-rich assays (Table 5). On the other hand, the assay for the GC-rich rs6275 (67% GC) worked well under standard conditions (Table 2), while the assay for rs1012944 (48% GC) required conditions otherwise used for the GC-rich sequences (Table 6). Therefore, proceed with all assays that developed low signal in both samples and NTCs as with AT-rich sequences, and with all assays that developed high signal in both samples and NTCs as with GC-rich sequences. If the problem has not been solved, you will have to re-design the primers. One very special case is described in Note 4.

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