Does anyone know what are the methods/techniques/ways to overcome slippage of repetitive DNA sequences such as microsatellites during PCR amplification?
You can try ISSR (Inter simple sequence repeat) PCR to avoid microsatellite slippage...
Design primers that align/anneal to regions that do not contain repetitive sequences, especially at the 3' ends. You may need to make longer primers, say 50-90 bp in order to do that.
Thanks Gary. But I am actually interested in the microsatellites regions. I am looking at microsatellite instability in formalin-fixed samples of cancer tissues. So that means I have to amplify across the microsatellite regions. Would designing longer primers in this instance be able to overcome the slippage which occurs in the microsatellite regions?
If your target region having microsatellites have more than 60% GC content, Mgcl2 or DMSO can be used for better amplification of the region. Commercially available GC rich kit can also be used.
Okwy, when you say you have to amplify across the microsatilite regions, are the primers annealing within the microsatilite region or outside it? If the primers can anneal outside the microsatilite region then I would make them so that they anneal to non-repetitive sequences and their 3' ends are first extended for 500-1000 bases before reaching the microsatilite region.
From Wikipedia (https://en.wikipedia.org/wiki/Microsatellite):
Creation of microsatellite primers
If searching for microsatellite markers in specific regions of a genome, for example within a particular exon of a gene, primers can be designed manually. This involves searching the genomic DNA sequence for microsatellite repeats, which can be done by eye or by using automated tools such as repeat masker (http://www.repeatmasker.org/). Once the potentially useful microsatellites are determined (removing non-useful ones such as those with random inserts within the repeat region), the flanking sequences can be used to design oligonucleotide primers which will amplify the specific microsatellite repeat in a PCR reaction.
Thanks. But I already know the microsatellites I am interested in. I just want to find out if they are elongated or shortened in cancer. Is your suggested primer design still applicable in this case? The primers which I am presently using are 20- 25 bp long and are sited at some distances from the microsatellite repeats
Yes, my primer design is applicable. And your primers are fairly short, primers 50-70 bp will be a lot more specific. The longer the non-repetitive sequence the PCR products have to anneal to the less slippage. So if you are amplifying 200 bp of repetitive sequence, try having 600 bp of non-repetitive sequence (300 bp on both sides of the repetitive seq.) for a PCR product of 800 bp.
HI Henry, have you managed to amplify DNA sequence with repeats? I am also trying to amply a DNA with multiple repeats.
No, not yet. By mid-May I should be doing that. What about you? What have you got?
Hi Henry,
Can you give me an update on how you have overcome this problem? I am in the same situation.
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