BISC 219: Mod 2 Lab 8

PCR Reaction Cleanup and Sequencing

In order for our sequencing reactions to work properly we need to remove the components of our PCR reaction (like dNTPs, Taq and salts) that might prevent the sequencing from working properly.

We will be using the Qiagen QIAquick PCR Purification Kit (catalog #28104). We will follow the instructions as they are in the Qiagen protocol book.


Sequencing:
BACKGROUND: Sequencing a portion of our genes of interest will provide us with direct evidence of the nature of the mutation (i.e: point mutation or deletion) in the mutant worms, as well as if RNAi has any affect on DNA sequence. It will help us make conclusions about where in the DNA to RNA to protein pathway RNAi actually works. This knowledge will be useful to establish a link with the type of functional defect present in the protein produced by the mutated gene.

There are three major steps in a sequencing reaction, which are repeated for 25 cycles in a thermocycler:

1. Denaturation at 94°C: During the denaturation, the double strand melts open to single stranded DNA and all enzymatic reactions stop, including the extension from the previous cycle.
   
2. Annealing at 50°C: In each sequencing reaction, a specific primer is required. This primer is a short sequence of bases complementary to a region of the plasmid upstream of the target gene. The primer will anneal and stay, we hope, only at one position on the single stranded DNA template. In sequencing reactions there is only one strand copied whereas, in PCR, two primers are used so both strands of the DNA template are copied. The primer jiggles around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that small section of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
   
3. Extension at 60°C: Sixty degrees is the ideal working temperature for polymerase activity in sequencing even though, normally, it is 72 °C. Because the growing strand must incorporate ddNTP's which are chemically modified with a fluorescent label, the temperature is lowered so it has time to incorporate the 'strange' molecules. Well designed primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, come loose again and don't give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3'side (adding dNTP's or ddNTP's from 5' to 3', reading from the template from 3' to 5' side, bases are added complementary to the template). When a ddNTP is incorporated, the extension reaction stops because a ddNTP contains a H-atom on the 3rd carbon atom (dNTP's contain a OH-atom on that position). Since the ddNTP's are fluorescently labeled, it is possible to detect the color of the last base of this fragment on an automated sequencer.
   

Because only one primer is used, only one strand is copied during sequencing, there is a linear (not log as in PCR) increase of the number of copies of one strand of the gene. Therefore, there has to be a large number of copies of the gene in the starting mixture for sequencing. If there are 1000 copies of the wanted gene before the cycling starts, after one cycle there will be 2000 copies: the 1000 original templates and 1000 complementary strands with each one fluorescent label on the last base. After two cycles, there will be 2000 complementary strands, three cycles will result in 3000 complementary strands and so on.

RNAi Schedule of Experiments
RNAi General Information
Media Recipes
Lab 4: Picking your gene to RNAi
Lab 5: Plasmid DNA isolation and transformation
Lab 6: Induction of RNAi plasmid and C. elegans feeding
Lab 7: Single Worm PCR and Agarose Gel Electrophoresis