DoingPCR.ppt-UniversityofVermont.ppt

3’ 5’ . Raise the temperature, and the strands elongate. 5’ 3’ 3’ 3’ 5’ 5’ 72° Elongation Cycle 2 3’ 5’ . Here is the final result…we now have four DNA molecules. In two cycles we went from one to four, so we are doubling the DNA with each cycle. This is why it is called the chain reaction, because each cycle builds on the previous cycle, a kind of chain reaction. Thus, the name of Polymerase Chain Reaction, or PCR. 5’ 3’ 3’ 3’ 5’ 5’ 72° Elongation Result of Cycle 2 3’ 5’ . Notice that you still have an extra overhang of DNA on each segment that you have amplified. Let’s look at the 4 DNA molecules. We have 2 of the A types, so we have not increased their number (we had 2 A’s at the end of Cycle 1), and we now have two molecules with unwanted DNA only at one end, and it is a single stranded piece. Let’s call that the “B” type of DNA molecule. Notice that we don’t have any of our pure target DNA (no pure red strands). 5’ 3’ 3’ 3’ 5’ 5’ 72° Elongation Result of Cycle 2 3’ 5’ . Melt... 5’ 3’ 3’ 3’ 5’ 5’ Cycle 3 begins 3’ 5’ . Anneal and elongate to get this result….We now have 8 DNA ds molecules. Because of space Limitations, sometimes a double stranded piece of DNA will show up as a wide line (sorry). Let’s look at the kind of strands present. We have 2 A type (no increase), 4 B’s (so they have doubled), and a new kind of segment, one that is double stranded pure target. That is our goal! Call these two DNA molecules the Targets. 5’ 3’ 3’ 3’ 5’ 5’ Results of Cycle 3 3’ 5’ . The two targets will replicate each cycle, so we have 2 here at the end of Cycle 3, and will have 4 at the end of Cycle 4, 8 at the end of cycle 5, and so on. These two strands will be the ancestor of millions of strands after 35 or so cycles. And, all of those strands will be the exact sequence you wanted to amplify. But, what happens to the other 6 strands here? Let’s put our two “target” fragments off-screen (but whatever is off-screen will dou