BioCurious Message Board › Explanation of Terms
|A former member||
I tried to explain the major terms of today's presentation by John Schloendorn. I hope this helps...
DNA is a string of molecules (well, two strings, really), which are differentiated by four bases (organic compounds). These four bases are Adenine (A), Cytosine (C), Guanine (G) and Thymine (T).
DNA is made of two strings of these molecules, but we only need to know one string to know both. This is due to Watson-Crick base pairing. This means that A-T and C-G create weak bonds between each other, when they are close to each other. DNA exploits this to create a more stable molecule consisting of two strings, ie:
CATGAC -> template string
GTACTG -> complement string
This is called complementarity and it means that if we know one string, we already know the other by creating the complement.
What is special about DNA is that different proteins in the cell recognize different subsequences of DNA and perform some action when those subsequences are found.
Below is (from memory) the gene we created
EcoRI - Interleukin2 - TEV - His(6) - BamHI
Here are the actions that different proteins take on these subsequences:
EcoRI and BamHI (Restriction Enzyme sites) are recognized by enzymes called Restriction Enzymes. A funky name that doesn't immediately connote their function. Restrictions enzymes cut DNA in a very specific way. There is then another set of proteins that can repair this type of cut. Sounds useless until you realize that lab conditions allow us to control when the cutting enzymes and repairing enzymes are exposed to our gene.
We introduce the cutting enzymes when we want to release our gene from whatever DNA it is in. We then isolate our gene and put it into a new mixture with the DNA we want our gene to be in. We introduce the cutting enzymes again, to open up the DNA. We then neutralize the cutting enzymes and add the repair enzymes and...wha-la, our gene is in the DNA we want.
Interleukin2 (gene of interest) Simply put, this is the protein we want. John explained how this gene is used by the cell to create proteins. In short gene -> mRNA -> protein
TEV is similar to a restriction enzyme site. The difference is that the recognition and cutting are performed on the amino acid sequence (aka the protein), instead of the nucleic acid sequence (aka DNA). Just to be clear, proteins other than restriction enzymes perform this cutting.
TEV allows us to separate our interleukin2 protein from the next subsequence, the hexa-histidine sequence.
His(6) aka hexa-histidine aka Histidine Tag. Histidine is one of the naturally occuring amino acids which make up proteins. The important thing about this sequence is the occurence of six of the same amino acids in a row. The chance of this happening randomly in a protein sequence is extremely small. Therefore, we know that any protein that contains this sequence is the one we created.
We use the chemical properties of histidine to attract our interleukin2-his(6) protein to another object (John talked of beads being the attractor) which we then remove from the rest of the cell or other stuff in solution. In short, it allows us to isolate the protein, after it's been created by the E. coli cell.
Briefly, and in chronological order:
EcoRI/BamHI: move our entire DNA sequence out of and into larger DNA molecules
Interleukin2: the protein that we want
His(6): isolate our protein from other proteins in the cell, after the sequence has been translated into protein
TEV: separate the interleukin2 from the histidine tag, after isolating our protein
|A former member||
Very well said. . . . .
However as a nitpicker, I must mention that when those nucleotide bases are strung together they are then a single long molecule. Due to the weakness of the double strand bonding which can be undone by heating to 95 degrees C, they are still considered two molecules despite how well they fit together at low enough temperatures.