I'm a great believer in luck, and I find
the harder I work the more I have of it.
Thomas Jefferson

Day 1: Primer design

Assignments Due

Discussion Topics

Experimental Procedures


Polymerase Chain Reaction

Polymerase chain reaction (PCR®) produces many copies of segments of DNA. The reproduction relies on the ability of DNA polymerase to make double stranded DNA. If a shorter, complementary segment of single stranded DNA (primer) is annealed to a longer single strand of DNA (template), the polymerase will extend the 3' end of the shorter segment to create double stranded DNA. Using two primers that are complementary to opposite strands, the DNA between and including the two primers is amplified. One cycle of amplification of DNA consists of three steps:

  1. Denature the double strands by melting (90-94°C)
  2. Anneal the primers to the template DNA at a reduced temperature (55-70°C)
  3. Synthesize (extend) the complementary DNA (70-75°C) with a DNA polymerase

Usually, 30 cycles are run yielding a near doubling of the number of DNA copies in each cycle. Actual efficiencies approach 1.8 fold increase in copy number/cycle.

Originally, DNA polymerase had to be added after each denaturation step but a thermal stable polymerase isolated from Thermus aquaticus (Taq) eliminated this tedious and expensive step. DNA fragments of fewer than 10,000 base pairs can be amplified with Taq. Longer fragments tend to fail due to mismatch errors that stall the enzyme. Several other thermal stable enzymes have been isolated that can survive the heating cycles. Each has varying efficiencies of proofreading and exonuclease activity for the correction of mismatch errors. A combination of two enzymes achieves amplification of longer lengths of DNA, up to 30-40 kb.

The efficiency of the PCR reaction necessitates scrupulous attention to keeping solutions clean and free from contamination. Some laboratories devote separate areas and equipment as PCR positive and PCR negative to prevent possible cross contamination. The preparation of target DNA should never occur in the same space as the preparation of the PCR reaction. (This guideline is not possible in the teaching lab making the negative control very important in our experiment.) Positive displacement pipettes or special filtered pipette tips are used to prevent contamination. For diagnostic and very sensitive procedures, reactions are set up in a laminar flow hood and all non-DNA solutions and containers are exposed to UV light to destroy errant DNA. Care must be taken to prevent contamination of the stock solutions. PCR on 5 µl of a 1 x 10(4)-fold dilution of the plasmid preparation in this experiment can produce a visible band on the agarose gel.

The manufacturer's protocol recommends that 1 x 10(5)-1 x 10(6) target molecules be placed in the reaction mix. Consider the following relationships as a guideline for amplification of single copy genes:

Primer design is a critical component of PCR -- ideally, a primer pair will anneal specifically to the target sequence and amplify just that region. Online tools, such as Primer3 (Center for Genome Research, Whitehead Institute for Biomedical Research), and software packages, such as the Wisconsin Package from Genetics Computer Group (GCG), help you design effective primers for PCR. However, there are several simple guidelines you can follow to design good primers "by hand."

For some useful information, see these guides from Invitrogen and Promega.
***Just for fun, check out Bio-Rad's Scientists for Better PCR***

Experimental Procedures

Primer design for PCR

You will work in teams and use online tools to design a set of primers for qPCR of a gene in Arabidopsis.  You will choose a set of primers for normalization from the references we give you in class.

  1. Download the sequence of your gene. To obtain the sequence, visit the Salk Institute and type At_g_____ into the query box. Select the highlighted green bars and then click on the [Seq] link to pull up the gene sequence. The default is to display the sequence with introns removed, which is great because qPCR uses cDNA as template. Locate the full-length version of the gene and copy the sequence.
  2. BLAST the sequence against the Arabidopsis genome to map areas with homology to other targets. Select Arabidopsis thaliana and paste the gene sequence into the FASTA sequence box. We will use a database of cDNA sequences from Arabidopsis to identify homologous sequences. How will you map these to your original sequence?
  3. Select a target region and model secondary structure. At mfold enter your sequence into a DNA form and identify regions that could self anneal after denaturation.
  4. Design primers using Primer3.   A target amplicon will be between 75-200 bp. We will discuss primer design parameters to use as settings at Primer3 website.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 23 July 2003
Updated 10 March 2015