We are performing in the Laboratory the PCR of the gp60 gene of Cryptosporidium. In many cases it does not work and some colleagues have told me that in some genes like this, which have a microsatellite region with a variable number of trinucleotide repeats (TCA, TCG, or TCT) coding for the amino acid serine, the TaqDNA polymerase skate, giving multiple products with different sizes. We use a nested PCR protocol in two steps with primary PCR primers AL3531 and AL3535 and secondary primers AL3532 and AL3534.
Could anyone help me solve this to obtain one band?
I forgot to tell you that if your problem is the stutter bands you can try the PCR amplification with a faster DNA Polymerase that will help in reducing them.
One of the reasons of getting multiple bands is if your primers are non-specific. As you mentioned your target gene has microsatelite sequence, so please check whether the primers are designed from that region. If the answer is yes, then you need to redesign primers avoiding microsatellite seq and if the answer is no, then try to adjust PCR conditions like temperatrure (gradient PCR will help to fix the temp), primer concentration, MgCl2 concentration etc. It is better to do one varibale at a time.
Multiple bands are unavoidable when you are amplifying microsatellites. Not a single trick from the book will prevent these ... nested PCR included, may even enhance the problem ... You just have to live with them and determine which of these multible bands you should assign!
If the region you are amplifying is a microsatellite, then is normal to see in the analysis what is called as "stutter bands" generated by DNA slippage during PCR amplification. You can recognize them because, If your microsatellite sequence is composed by a trinucleotide repetition unit, then between the stutter bands the size difference will be multiiple of three (i.e.: 120bp, 123bp, 126bp...). When analyzing SSR markers it can be also detected +A bands. When this happens usually you only see the +A band corresponding to the band of the allele, that means that you will see a band 1bp bigger. But if the pattern of the SSR that it is been analyzed presents a lot of stutter bands, the +A bands of each stutter one may havealso been amplified, and in this case you will see your allele band, the stutter bands and the +A bands of all the other bands. To avoid the presence of +A bands a long final extension cycle can be added in your PCR program (for example, 72ºC for 30 min or 60 min or 90 min; you have to optimize temperature and duration of this last cycle to your primers) May I ask how are you visualizing the PCR products?
I forgot to tell you that if your problem is the stutter bands you can try the PCR amplification with a faster DNA Polymerase that will help in reducing them.
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