Chromosome Strand, Gene Direction, Restriction Enzyme Recognition

Question

[Kate]

Hello, I want to design my new experiment and I am a little bit confused.

1. I have genome sequence frm sequencing project
2. In the genome sequence are the genes of my interest
3. I do RNA isolation, cdNA synthesis and then restriction enzyme cutting

My problem is:

In the genome I have genes in opposite directions and I do not know how to specify 5'-3' strand. I thoutght it would be good to find codon start and codon stop, and then sequence begiining from atg is 5'. I think it is important because restriction enzyme recognizes 5'3; sequence so that sequence should be checked for restriction enzyme sites, If I check different strand I can find different enzyme sites. Is that correct?

[Pierre Lindenbaum]

most restriction enzymes (type II) digest a palindromic site (e.g GAATTC for EcoR1), so I don't think you need to care about the strand of your genes.

[Damian Kao]

Depending on how your genome is annotated, you might not get a clear cut start codon and end codon. You might have picked up UTRs or have wrong splice junctions. What is your goal for the restriction? Are you trying to figure out if the gene of interest is the correct size in your annotations?

Answer 1

[Pablacious]

I'm not sure if I'm understanding all of it well, but I have a few hints that I guess you could use:

For the finding restriction sites you can use the EMBOSS package, which is available to use either through web interfaces or command line, what ever you prefer. You can access it for direct web use in:

http://emboss.bioinformatics.nl/cgi-bin/emboss/restrict

Basically you submit a sequence and it will try to find restriction sites for you. You can specify I think which enzymes to use. It will look for the restriction sites in both directions (I'm almost sure, I used this last time several years ago), so you don't need to worry about that yourself.

Regarding finding your genes, I'm guessing for what you write that you haven't actually defined the positions of them yet in your sequence. For this you classically need a gene marker (such as Glimmer3, Critica, there are probably some newer ones, haven't been in genome annotation field for a little while now) program/algorithm. You would normally use Glimmer in the command line, as there are many ways of training it, and it really depends on the data you have. Assuming that you are not familiarized with running unix programs, in the case of Glimmer, you can use an already trained bacterial version on:

http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi

Glimmer will tell you where you probably have genes in your sequence (where they end and where they start). The business is slightly more tricky than just finding start and stop codons ;-).

These two tools of course only make sense for microbial genomes. Good luck ;-).

Answer 2

[Kate]

Thank you for your answer.I have bacterial genome sequence. I try to find out where the beginning and end of gene is by comparing to databases of homologic genes. I still think that restriction recognition site is important even it is palindromic-because still the reverse strand should be in correct 5'-3' direction. I need to have restrction sites for cDNA to project digestions.

[Larry_Parnell]

Kate, it is nice that you thank those who have responded to your question. This statement from you, however, is a comment to the above and not an answer itself. Please delete this and add it as a comment under your question.