We are trying to purchase a suitable set of primers and restriction enzymes for the qualitative diagnosis of of chronic myeloid leukaemia using PCR and restriction fragment length poly,orphism. Can any one help us with the details of primers, their PCR conditions and restriction enzymes that we can use?
Hello Sir!
If you want to design a set of primers, I hope I can provide you a newly designed primer with the specifications required.
Restriction enzymes and PCR conditions will depend on the primers used and the resulting amplicon size. Hence, the 1st question is required.
Article Serial monitoring of T315I BCR-ABL mutation by Invader assay...
follow the link.
Hi everyone
Anazoeze Jude Madu
The first and important thing is to know what enzyme will you use and is it provided in your country. About RE, in wild or mutant type, when you have the mutant gene, the enzyme recognizes your SNP and will cuted and your wild type won't cut, or vice versa.
For Primer designing, the length for the PCR product is important, because when your enzyme does not recognize and does not cut the PCR product, you'll see one band. It's clear so far, in the next state, the enzyme does recognize the cutting site (in mutant or wild type, it depends on the polymorphism), you'll see two bands, and at the first step in designing primers, you must in such way designed your primers that after recognition and digestion the PCR product with the enzyme you see two bands in different length (for example your PCR product length (amplicon) is 1400bp, the enzyme recognize the SNP at 900bp and after digestion, you see 900bp and 500bp bands), so these two bands are separable in electrophoresis and it is homozygous. If the SNP is hetero, you'll see 3 bands, 1400bp, 900bp, and 700bp.
So, if after recognition and digestion of the enzyme, your bands have the same length, you'll cant find out homozygous or hetero, because in electrophoresis two bands fall on each other.
With regards
Farshid
- Badges
- Science method