Transgenetic method?

Question

When creating a transgenetic organism using cDNA (DNA compliment) Is it necessary to include the introns and promoters? Or do I rely on the organism's own DNA regulatory sequences? Basically, I would like to know if transformation of a gene is possible with the sequence from cDNA alone, or if the genomic DNA sequence is needed?

Answer 1

It depends. If you are transforming a non-native sequence you are better off using the cDNA, otherwise you will have to engineer the genomic DNA to make sure it has the splice sites that the organism you are transforming will be able to splice properly. Also usually when you make a transgenic you provide the promoter you want to use. You should not transform a cDNA without some type of promoter in your vector.

In short yes you can transform with cDNA as long as you put it in an expression vector.

Answer 2

cDNA will differ from genomic DNA because cDNA is DNA copied from the mRNA's present in your sample rather than the complete genomic DNA. Genomic clones represent a random sample of all the DNA sequences found in the organisms genome and will probably be the same no matter what tissue or cell is extracted. Are you cloning a Eukaryotic organism? another good question. .. They contain not only a large amount of repeat sequences but introns, gene regulatory regions, and spacer DNA, and codes for proteins... cDNA contains only the coding sequences and ONly those for genes that have been transcribed into mRNA... IN THE TISSUE FROM WHICH IT WAS extracted... differnt tissues and cells have distinct mRNA molecules... so a differnt cDNA library will be obtained for each type of tissue....The advantage of cDNA clones is that they contain the uninterrrupted coding sequence of the gene,... IF the aim of cloning the gene is to find the amino acid sequence of the protein from the DNA or to produce the protein in bulk by expressing the cloned gene in a bacteria or yeast cell.. this works great... Genomic clones contain introns and exons and will include the necessary regulatory sequences that determine which genes are expressed in what tissue and how much protein will be made... genomic clones are what is used to peice together the individual nucleotide sequences...I would suggest .... extraction of DNA and then...and old fashion PCR...;-)