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 región 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 with primary PCR primers AL3531 and AL3535 and secondary primers AL3532 and AL3534 Does anyone can help me solve this to obtain one band?
Dear Pilar Goni,
Yes its true amplifying this gene target is difficult from our experience but nevertheless not all cases that result into multiple bands as shown in the attached gel.
Dear
amplifying this gene target is difficult but in many isolates of this parasite you could amplify this region. However it is better that you consult with a molecular prasitologist
hi dear, Your test is likely to suffer contamination. Or to choose primers from another part
i'm agree with praveen, try to make a primer BLAST and see if anneal in the pseudogene if you are making a nested PCR check the dilutions for the fisrt product all the products remaining can create noise in the results
exitos!
Thank you very much for your suggestions. I comment in more detail, we always do the nested PCR in two steps and there are some samples with fewer repetitions which work out well. But lately, those with more repetitions are the most abundant and we can not change the amplification area because precisely these repetitions serve to characterize the isolate of Cryptosporidium
I'm sorry I do not know a lot about molecular biology. And how to arrange the rules and change events in the genes. Mutagens. And how to use primers. To get the packages. And causing a breach in the order of nitrogenous bases. But according to knowledge. Downright breach of nuclides in the order of the error occurs. Change and possible break in the bar DNA.
GP60 gene PCR
Try to use this protocol (I used and works well)
A fragment of the 60 kDa glycoprotein (GP60) gene was amplified by nested-PCR (PENG et al.,2003; GLABERMAN et al., 2002) using primers 5’-ATAGTCTCCGCTGTATTC-3’ and 5’-GCAGAGGAACCAGCATC-3’ (primary PCR) and 5’-TCCGCTGTATTCTCAGCC-3’ and 5’-GAGATATATCTTGGTGCG-3’ (secondary PCR). These primers were designed based on sequences conserved among all known C. parvum GP60 alleles.
Try to use an ABI 3500 Genetic Analyzer® (Applied Biosystems, USA) or other similar.
Remember that double bands in agarose gell may represent the presence of multiples genotypes in the same sample.
I used the exact same primers for the gp60 and have not experienced any issues with by products. However, I used HotStar master mix. You might want to dilute your DNA straight after extraction.
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