Gene cloning with unknown sequence - (Mar/29/2014 )
I need help with a project, so if anyone has an idea that could help me to start off, I will be gratefull.
My question is, how to clone a gene, from unsequencized organism, if you know the sequences of orthologue genes, that show high homology in nucleotids with unknown gene? Length of unknown gene is about 5 bp. Thank you in advance
5 bp is very short??
By PCR for example , but 5 bps?
Can you give more details?
That is the theme for a paper, and all they said to us. My idea was to try making complementary DNA based on known (and highly homologue) sequence that is given, but as you all say it is too short to hibridize only with my unknown sequence.. So i guess I need another approach :/
5 bp is not enough to make a gene - you could only express 1 amino acid (codon: ATG coding for methionine), with a couple of bases left over, not even enough for a stop codon. This means that it does not make a protein as these are polypeptide (i.e. 3 or more amino acids). Perhaps you mean 50 bp or 500 bp or even 5000 bp?
Anyway to answer your question - you could design consensus primer sequences for the gene based on the sequences of related organisms, then attempt to amplify the gene from those. To get the exact sequence you would then need to sequence the DNA you have amplified and then do RACE to get the ends correct.
Thank you very much for the answer, i went over the assigment again and realized i was reading 5 bp instead of 5 kp, as it is written. Sorry for the trouble, I am embarrassed right now, for not thinking.
In that case - see my answer above.