Bacterial Artificial Chromosomes (BAC's)
Okay, so my work experience at a local genetics lab begins on Monday, and i shall be bringing you a diary of sorts concerning my time there. So far I've been informed that I will be constructing DNA libraries using BAC's. So i thought i's explain a little about what they are and how we can get the complete genomic sequence of an organism from them.
So BAC's are a construct of DNA, as for DNA to be taken up and cloned by an organism some kind of DNA vector (carrier) must be used. Escherichia coli is the most commonly used organism for this technique, but other organisms, such as Saccharomyces cerevisiae (yeast) can be used.
From these organisms a fertility (F.) Plasmid (usually used to swap resistance genes with other individuals, these are important as they promote the equal sharing of plasmids between progeny) is opened up using enzymes called restriction endonucleases. These will cut the DNA at a certain designated sequence, called the restriction site. (For trivia purposes this site is a palindrome, rather like the word 'racecar' the DNA will read the same on both sides, if you follow the 3 prime to 5 prime direction of the individual strand (they run in opposite directions, anti parallel))
The same endonuclease will be used to excise a gene from the organism you desire to sequence, such as a length of DNA from a human, so the ends of the components will have the same, complimentary sequence, and when mixed together may join together. They are mixed with another enzyme called DNA ligase.
If the two components, the organism's DNA and the opened F-plasmid, join together a recombinant plasmid is formed. This is the BAC, (some plasmids will seal up again and some of the organisms DNA will just join together as a loop, due to the nature of the palindrome).
Induced electrical fields are used to get the plasmids back into the bacterium (a process called electroporation), few bacteria will successfully take up the gene in the recombinant plasmid. Because the inserted gene of interest often disrupts a gene which has influence on the colour of the culture, colonies that have taken up the gene have no colour pigment and are white, and therefore are easy to spot.
These BAC's are used then to amplify the DNA and the sequences taken are then orded in silico (Using a computer program) so the sequence of the Genome can be determined.
As for the specific lab processes pertaining to this procedure I will give you a post on that after I have done it. Hopefully they may let me take a few photos of the machines if I ask nicely!
