Dave Gray's Build-A-Gene Class Notes - Session 1
In session 1, we amplified three items:
- DNA Vector Template - (2070 base pairs) I believe this stretch of DNA contains the bulk of the "vector" including a marker and terminator. The terminator will wind being just after the emGFP coding sequence on the vector and will terminate the production of RNA. The vector template also includes a marker segment. (Need more info on how this will be used.)
- Promoter Template - (500 base pairs?) I believe this includes both the Promoter and the RBS (Ribosomal Binding Site). The Promoter is where RNA polymerase binds to the DNA just before the start of the gene to build RNA. The RBS defines where protein translation from the RNA begins. A ribosome links amino acids together in the order specified by messenger RNA.
- Control - To verify that our technique was sound and that we didn't inadvertently pollute our product with unwanted DNA.
Each of these are mixed with appropriate "primers" and PCR Master mix and then placed into the PCR machine.
The "primer" is a short strand of DNA (an oligonucleotide) that the enzymes can build on to replicate the entire strand we want. It is the starting point of the new strand. It contains a combination of nucleotides that will allow it to uniquely bind to the right starting point because the complementary sequence to the primer appears only one place in the DNA.
The master mix contains the buffer, enzyme and nucleotides. The buffer is a solution with the correct salt and PH levels to support replication. The enzyme does the work of attaching nucleotides to the DNA template to carry out replication.
The PCR machine runs through a number of cycles of heating and cooling. When it heats the mixture to near the boiling point of water, the DNA "denatures" (splits apart). As the mixture cools, the primers "anneal" to the DNA. Then the enzymes attach nucleotides to the primers to extend the complementary DNA strand. With each cycle of heating and cooling, the amount of DNA in the mix doubles, thus growing geometrically.
Note: As I understand it, the bonds that hold the two strands of DNA together are hydrogen bonds. This is the same bond that causes water molecules to stick together. Is that why the strands denature at approximately the boiling point of water - this is the right amount of energy to break hydrogen bonds?
All of this points out that we have to think of the DNA we are producing at three levels with three "starting points". The first level occurs during amplification. There the primers specify the starting point for replication of the DNA. By including a primer in the "forward" direction for one side of the DNA strand and a primer in the "reverse" direction for its complement, we are able to precisely replicate only the portion of the DNA we are interested in.
The second starting point is the promoter. It starts the transcription to RNA from DNA, which stops when it encounters the terminator. The promoter can be strong, weak or operate only in the presence of certain chemicals.
The third starting point begins with the Ribosomal Binding Site and is the point where translation of the RNA to a protein begins assembling amino acids.