Assignment: After listening to the in-class explanation of PCR, reading your background information and studying the diagrams provided, answer the following questions.

1. Review and briefly explain the steps involved in normal DNA replication within a cell before mitosis.

1. DNA is "unzipped" by an enzyme present in the nucleus.
   
   
2. DNA polymerase (an enzyme) travels down each separated strand, bringing in complementary nucleotides to build a new strand.
   
   
3. The sugar-phosphate bonds are formed along each new DNA strand.
   
   

2. What is the function of each of the following in a PCR reaction?

• Heat: Breaks the hydrogen bonds between the strands of DNA, separating the strands
  
  
• Primer: A small, single-stranded piece of DNA which attaches to a singleDNA strand and provides an anchor for the Taq polymerase to extend thenew DNA strand it is building
  
  
• Taq polymerase: A heat resistant enzyme which builds a new DNA strand from the end of a primer attached to the template DNA strand.
  
  

3. Briefly describe what the thermocycler (PCR machine) does and what happens in each of the following three steps of Polymerase Chain Reaction:

• Denaturing: Increase heat to 95°C to separate the DNA strands.
  
  
• Annealing: Lower the temperature to allow the primers to hydrogen bond to their complementary sequences along the spearated DNA strands.
  
  
• Extension: Raise the temperature to the optimum temperature for theTaq polymerase to work (72°C) in building the new strand from the primer forward.
  
  

4. Suppose you start with one double-stranded molecule of DNA and you want to amplify this one DNA molecule by PCR. You add an excess of a two single-stranded primers, each of which will anneal to the DNA molecule in only one place, copying the segment of DNA between them. Draw representations of the DNA and primers in each of 3 cycles. Label the template DNA, the primers, and the newly synthesized DNA strands. Show the direction of DNA synthesis on each strand.

How many molecules of double-stranded DNA will you have after three cycles? 8 After six cycles? 64 After 100 cycles? 1.27 x 1030 (1 pt. extra credit)

5. Compare the normal process of DNA replication within a cell to the artificial replication of DNA molecules in a PCR machine. Describe what is different between these two processes.

Using heat to separate the DNA strands in PCR instead of using a enzyme in DNA replication; needing a primer to privide a "hook" for the Taq polymerase to start building a new DNA strand from; needing a heat resistant polymerase so that the enzyme isn't destroyed when heat is used over and over to separate the strands, which isn't a problem in normal DNA replication.

6. What kinds of changes to chromosomes would cause a "polymorphism" to appear in the DNA profiles of different individuals from a population?

A mutation or rearrangement could cause a primer site to be lost, causing a band to disappear.

An insertion or a deletion between primer sites would cause the band to migrate a shorter or longer distance, respectively.

How would a deletion in the region between primer sites of one individual cause a band to run closer to the wells in a gel?

It would shorten the fragment amplified.

Science Education Connection Department of Biochemistry The University of Arizona May 1, 1997 warder@u.arizona.edu http://biology.arizona.edu/sciconn/lessons/alongi/ All contents copyright ©1997. All rights reserved.