- PCR PreLab
Before completing this Prelab, we ask that you (please) fill out thisshort, anonymous survey asking your opinion of the Bio 121 Prelabs, you will be returned to this page when you finish .

LABORATORY 10. The Polymerase Chain Reaction

Textbook sections:
19.2 Analyzing DNA: Did Our Ancestors Mate With Neanderthals? (p. 409)
The Polymerase Chain Reaction (PCR) (p. 410)

After reading the text material, you should be able to:
1. Give at least two reasons that scientists might want to determine the sequence of
a gene.
2. Explain why the ability to make many copies of a gene is important.

After completing this tutorial, you should be able to
1. Name the three steps that constitute one PCR cycle and give a detailed description
of each.
2. Discuss the relevance of Taq polymerase to PCR.
3. Predict the number of DNA molecules produced after a certain number of PCR
cycles.

Key terms and Concepts:
  • DNA polymerase - An enzyme that synthesizes a growing strand of DNA by adding bases that are complementary to a template strand.
  • Deoxynucleoside triphosphate (dNTP) - A monomer that is the essential building block for making a new strand of DNA.
  • Nucleotide - A monomer that can be polymerized to form DNA or RNA.
  • Polymerase chain reaction (PCR) - A DNA synthesis reaction that takes place in a laboratory, in which a specific section of DNA is replicated over and over to amplify the number of copies of that sequence.
  • Primer - A short, single-stranded sequence of RNA or DNA that enables the start of replication of a DNA sequence that is synthesized from the 3’ end of the primer.
  • Taq polymerase - A heat-stable form of DNA polymerase derived from a bacterium that lives in hot springs.

Overview - The polymerase chain reaction, or PCR, is a powerful method for obtaining millions of duplicates of a DNA fragment from (theoretically) just one original copy. The reaction begins with a solution containing the DNA from a cell. The goal is to amplify a specific region, the target DNA.

A PCR ThermocyclerThe solution contains Taq polymerase. Taq is a type of heat-stable DNA polymerase derived from a species of bacteria living in hot springs. Because Taq polymerase continues to function normally at high temperatures, using it allows researchers to separate the DNA strands without destroying the polymerase.

The solution also contains primers—short, single-stranded DNA molecules—and plenty of deoxynucleoside triphosphates, or dNTPs.

The PCR Cycle
Let’s examine the basic PCR cycle, which is typically repeated about 35 times.
Temperature changes are automated by PCR machines.
  • In the first step, denaturation, the solution is first heated to nearly boiling—95ºC. The heat breaks the hydrogen bonds between the two DNA strands and allows them to separate. Despite the intense heat, the Taq polymerase remains active.
  • In the second step, primer annealing, the temperature is dropped to around 60ºC. The exact temperature depends on the length and base composition of the primers. At this relatively low temperature, the primers can form hydrogen bonds with the single-stranded DNA. Two primer types are created, each one complementary in sequence to one of the two ends of the target DNA. To make the primers, the sequences at the ends of the target DNA must be known.
  • In the third and final step, extension, the temperature is increased to 72ºC. This is the optimal temperature at which Taq polymerase functions. The primers are essential in this process, because they provide free 3’ hydroxyl groups, to which the polymerase can add additional dNTPs. Each new dNTP that joins the growing strand is complementary to the nucleotide in the opposite strand.

At the end of this first cycle, there are two DNA copies instead of the one original copy. In
cycle 2 the process repeats, so that there are now four double-stranded copies. The process continues, doubling the number of target DNA copies with each cycle. Doubling occurs because each newly synthesized segment of DNA serves as a template in the subsequent cycle. Notice that more and more of the fragments consist of just the target DNA.

The Number of DNA Molecules per Cycle
Roll your cursor over the histogram bars in the graph in the animation below to find out how many target DNA molecules are produced in each cycle. You can see how the exponential increase in copies with each cycle results in an enormous number of copies by the end of the 35th cycle. The general formula for the number of DNA strands created by PCR is 2n where n = the # of PCR cycles.

The PCR "product" (DNA) can be used for a broad variety of experiments and analyses. Some examples are discussed below.

  • Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her DNA with a given sample, such as blood from a crime scene can be genetically compared to blood from a suspect. The sample may contain only a tiny amount of DNA, obtained from a source such as blood, semen, saliva, hair, or other organic material, so PCR is used to increase the amount.
  • Paternity testing
  • Detection of hereditary diseases - The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR. Each gene in question can easily be amplified through PCR by using the appropriate primers and then sequenced to detect mutations.
  • Viral diseases can be detected using PCR through amplification of the viral DNA. This analysis is possible right after infection, which can be from several days to several months before actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.
  • Cloning a gene, not to be confused with cloning a whole organism, describes the process of isolating a gene from one organism and then inserting it into another organism (now termed a genetically modified organism (GMO)). PCR is often used to amplify the gene, which can then be inserted into a vector (a vector is a piece of DNA which 'carries' the gene into the GMO) such as a plasmid (a circular DNA molecule). The DNA can then be transferred into an organism (the GMO) where the gene and its product can be studied more closely.
  • Analysis of ancient DNA - Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar.
  • Genotyping of specific mutations - Through the use of allele-specific PCR, one can easily determine which allele of a mutation or polymorphism an individual has. Here, one of the two primers is common, and would anneal a short distance away from the mutation, while the other anneals right on the variation. The 3' end of the allele-specific primer is modified, to only anneal if it matches one of the alleles. If the mutation of interest is a T or C single nucleotide polymorphism (T/C SNP), one would use two reactions, one containing a primer ending in T, and the other ending in C. The common primer would be the same. Following PCR, these two sets of reactions would be run out on an agarose gel, and the band pattern will tell you if the individual is homozygous T, homozygous C, or heterozygous T/C.
  • Comparison of gene expression - Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's expression. Ribonucleic acid (mRNA) can be reverse transcribed back into DNA (complementary DNA to be precise, known as cDNA), at which point traditional PCR can be applied to amplify the gene, this methodology is called RT-PCR. In most cases if there is more starting material (mRNA) of a gene then during PCR more copies of the gene will be generated. When the product of the PCR reaction are run on an agarose gel a band, corresponding to a gene, will appear larger on the gel (note that the band remains in the same location relative to the ladder, it will just appear fatter or brighter).

Questions: Answer the questions below, then submit them along with your name and Lab instructor.

1. Taq polymerase is a DNA polymerase from a particular organism, Thermus aquaticus, this specific DNA polymerase is used in PCR because....?

It is more specific for the PCR primers
It can extend the primers to a greater extent
It does not require ATP to polymerize DNA
It resists the heating that occurs in the PCR cycle

2. A general formula for the # of DNA copies/cycle produced during PCR would be...?

2n where n = the # of cycles
2n where n = the # of DNA copies
2n/2/2 where n = the # of cycles
2 X (n) where n = the # of cycles

3. The three steps of a PCR cycle are..?

Denature - Anneal - Extend
Denature - Extend - Anneal
Heat - Extend - Anneal
Heat - Extend - Polymerize
Denature - Polymerize - Anneal

4. Describe one reason why the ability to make many copies of a gene is important.

5. Which of the below is an example of a deoxynucleoside triphosphate (dNTP)?

A

B

C

6. The forces holding the two strands of DNA together, prior to denaturation by heat during a PCR reaction, are..?

A. Hydrogen bonds
B. Cysteine bridges
C. Nucleotide attractions
D. Ionic bonds
E. Covalent bonds
F. van der Waals forces

Name:
Section:

Submit your answers to obtain a printable receipt. Be sure to select your section from the drop-down box.