Cloning of an unknown gene - (Apr/13/2015 )
I would appreciate some help on my assingment, I have an idea but I would like to hear your opinion since I'm still a uni student and I'm sure lots of you have more experience than I have
Size of an unknown gene is 10kb.
Some parts of an unknown gene have high homology with sequenced orthologous genes.
What is necessary for cloning process? Describe cloning method in details.
So my idea was:
1. Searching for a location of orthologous genes in plasmid genome library using FISH and isolation of these genes from library clone using restriction digest with two enzymes (restriction sites are surrounding each of orthologous genes)
2. Subcloning of digested genes into expressive vectors
3. Choosing the vector in which unknown gene product is expressed (I'm not sure about this because the product of the gene is not given in assignment)
4. Isolation of analyzed gene
5. PCR amplification: a ) designing forward and reverse primers complementary to conserved sequences of orthologous genes.
Each of these primers will have restriction site (for one of two used enzymes) and hybridisation sequence complementary to 5' (in forward primer) and 3'(in reverse primer) end of the gene
b ) PCR reaction
6. Verification of PCR reaction using electrophoresis on agarose gel
7. Isolation of analyzed gene using PCR Purification Kit
8. Choosing a plasmid vector which capacity matches size of the gene and which has two restrictiond sites for each of used enzymes (and antibiotic resistance gene)
9. Restriction digest of PCR product and recipient plasmid
10. Electophoresis of digest products on agarose gel and cutting off the bands which positions on the gel match the size of plasmid and analyzed gene represented on the ladder
11. DNA ligation of analyzed gene and recipient plasmid
12. Transformation of a host cell (E. coli)
13. Plantation (I'm not sure I'm using the right term here xD) of host cells onto medium containing the antibiotic
14. Overnight incubation
15. Identification of bacterial colonies
16. DNA purification (separation of plasmid DNA from bacterial culture)
17. Repeat steps 9 and 10
What steps should I change? What sould I add and what should I remove?
I would approach this primarily by identifying and sequencing the surrounding context of the gene. I would use inverse pcr from the region I knew sequence for to locate surrounding sequence. You might need to do this more than once to encompass the entire gene. Once I had the entire gene sequence, I would either use PCR to amplify the entire gene, or (more likely now) recode the gene and send it for synthesis. Your approach will likely work, but it is relatively old-school. An even more modern approach would be (depending on the complexity of the organism genome) to simply sequence the entire genome, assemble, and find the sequence of your gene by inspection.
I forgot to mention that the genome of an organism which gene I have to clone is not sequenced. I only know sequences of orthologous genes. I've read about degenerate primers which are synthesized using softvers for sequence prediction based on homology between orthologous genes. Do you think it is a good alternative? In my assignment professor is not favouring sequencing because of its expense.
I suggested inverse PCR, which I still think is your best bet, assuming you know sequence for a portion of your gene. If you don't know sequence, then a search for homologous genes and an alignment to identify highly conserved regions will give you a head start on designing degenerate primers. Look for single-codon residues, such as methionine or tryptophan, where, if you know the AA sequence, you know the nucleotide sequence. Avoid six-codon AAs such as leucine in your region.
Back in the old days, when no full genomes were available (gasp!), one could search for orthologs by screening colonies containing a plasmid-based genome library (or cDNA library) with a DNA probe made from the known ortholog. For example, if Gene X had been cloned and sequenced for the rat, you could make a labeled probe out of a cloned fragment of rat Gene X and use it to search for the Gene X in the mouse. One would use the probe to screen circular Southern blots pulled from agar plates containing the library colonies. Hopefully, one colony would “light up” with the probe (we used 32-P back then!) on autoradiograms. This was very laborious for a large genome like rat or mouse, and sometimes ~100’s of plates had to be screened! Yecch! These days you can screen these library colonies by colony PCR, in 96 or even 384 well plates. It helps if you have qPCR equipment for this so you don’t have to run the PCR products on gels, which would be nearly as bad as hybridizing hundreds of southern blots. However, if you have a small genome size (a bacterium, protist, or yeast?) this could still be do-able. And if your genome is large, build your libraries in BACs (Bacterial Artificial Chromosomes: like very large plasmids that can hold very large inserts), instead of generic cloning plasmids. Still, even this is fairly old technology.
I’m guessing your gene of 10 kb is probably from a Eukaryote with a sizable genome, and probably has introns in it. You should probably go with PCR based on conserved regions of the aligned orthologs as already suggested, maybe getting it out in pieces rather than trying to get the whole 10 kb at once. If you can amplify and clone a piece in the beginning and a piece near the end, you can then use the sequence of the two pieces to design accurate (no degeneracies) long range primers to try to amplify the whole thing.
In addition to the primer design advice above (use of degenerate primers), try to put the most highly conserved sequence (with no degeneracies) at the 3’ end of the primer, and let the degeneracies reside closer to the 5’ end. Since the polymerase adds to the 3’ end, that is where you need to have the most solid primer-template match-up. Also, be very careful about PCR cross-contamination issues. Once we were using primers based on our lab’s rat clones to amplify a bovine DNA template. We got a product, sequenced it, and it was IDENTICAL to rat- meaning it was a contamination product. This was before the invention of filtered aerosol-barrier pipet tips (which have improved the contamination problem immensely!) You can see why I'm called Old Cloner!
Check out this list of techniques:
Hope this helps and good luck!