rDNA.

Hola! So it is Biology paper tomorrow. And here I am, as always, to rant about what I've read. Wel honestly, I haven;t finished reading yet but it's 2 in the morning and I can't stand it. Yet I must keep going as this is the last paper I'm really gonna pay attention to. So here goes.

I'm gonna write only about recombinant DNA technology for now as this is the topics that seems to stubborn to get into my brain to be engulfed!

First, isolating target gene. There are two ways in doing this. First, cutting the gene from a complete chromosome. Well, this is called DNA extraction. There are four steps for this. Cell disruption to expose the DNA, removing membrane lipids by using detergent since it can digest the membrane, removing proteins by adding a protease and precipitating DNA with ice-cold alcohol producing a pellet upon centrifugation. The pellet should be washed to remove unwanted cell. And the second way in isolating the gene is by producing cDNA through reverse transcription. mRNA of target gene is extracted from the cells where the gene is actively synthesizing protein. mRNA acts as template, synthesizing DNA by enzyme reverse transcriptase. cDNA produces is intron-less.

Then there are this thing called restriction enzyme. it is used to digest DNA. It produces fragments of various lengths, mostly cut in staggered way results in DNA fragments with unpaired base on the ends called sticky ends. This restriction enzyme is naturally found in bacteria that are resistant to bacteriophage infection.

Second step of recombinant DNA technology is inserting the gene int a vector. So here's a thing. There a lot of characteristics the vector must possess. First, it must possess marker genes, ampR and lacZ, must be able to carry foreign DNA into host cell, must be digested with the same restriction enzyme used to digest target DNA to produce same sequence of sticky ends. Vectors which are in the form of opened rings with sticky ends attract complementary bases of other sticky ends and form H bonds. The fragments will be annealed by DNA ligase. Some examples of vectors are bacterial plasmids which is able to carry 10 to 15 kb base pairs. There's also bacteriophage with capacity of 25kb.  It is a linear double helix DNA, a part of it which is not required for this process is removed and replaced with target gene. This process is called transfection. IT has high transformation efficiency which is about 1000 times better than plasmid vector. Then there are cosmids, a combo of bacteriophage and plasmid. It infect host cell phage-like and is able to carry up to 45 kb. Last but not least, YAC (yeast artificial chromosome). It has huge capacity of 2MB but low efficiency of transformation.

Third stage, introducing the rDNA to the host cell. So here goes, the host cell MUST :

• be able to accept foreign DNA
• be able to maintain structure of rDNA from generation to another
• be able to amplify the gene of rDNA

rDNA is introduced to host cell through transformation. Mixture of these are mixed with e.Coli in a medium containing CaCl2 as it induces the permeability of cell wall. Small number of bacteria take up the plasmids only a small fraction of these bacteria contain recombinant plasmids.

Last but not least! The coning of gene by host cell step. The host cell is cultured in a restriction medium containg ampicillin and sugar X-gal. The transformed e.Coli which takes up the plasmids will be able to grow and form colonies on the medium. Each time the bacteria divide, the inserted gene also replicates. Bacteria containing lacZ gene able to hydrolyze X-gal. this produces blue compound which makes the colony blue (empty). The white colonies (lacZ not functioning) cultured in large scale. Each time the bacterial cell divide, the recombinant plasmid also replicates producing multiple copies of bacteria.

Oh my oh my! It's almost 4!!!!! I'm freaking out!