PCR Biological Method Overview

PCR, or polymerase chain reaction, is a method to amplify a segment of DNA for analysis. Because it is such a powerful technique, there are a HUGE number of situations where PCR may be used. Some common reasons for using it are:

1. Microbiology: You need to know if your bacteria was transformed properly with your plasmid
2. Oncology: You need to know if a particular gene exists in your cancerous cells
3. Forensics: You need to know if a certain criminal’s DNA was present at the crime scene

The basic steps of PCR include:

1. Designing primers to designate a target DNA sequence to amplify
2. Mixing together the primers with the target DNA strand, polymerase enzyme, and deoxynucleotides
3. Running a thermocycler multiple times in “cycles” to repeatedly:
   • Separate DNA strands
   • Allow primers to anneal
   • Allow the polymerase to attach and synthesize a new DNA chain
4. Sequencing or Electrophoresis to prove that the PCR worked

In a nutshell, here is what Step 3, above, looks like:

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Ingredients in a PCR mixture – What every component does

In general, a PCR tube will contain the following items:

Complete Step-By-Step PCR Protocol

Materials

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Methods

1. For each PCR tube set up the following mixture of materials up to a 50 ul total volume on ice
   

   dNTP solution	200 uM for each dNTP
   Forward Primer	0.2 uM
   Reverse Primer	0.2 uM
   Target DNA	Less than 1000 ng
   Taq Polymerase	1.25 units per tube
   Nuclease Free Water	Q.s up to 50 ul
   Mineral Oil	Add a little bit on top of each tube if you don’t have a heated lid on the temperature cycler

2. Pipette each tube up and down several times, gently, so as not to add bubbles
3. Centrifuge each tube down for 1-2 seconds at 100 rcf to bring all contents to the bottom
4. Heat up the temperature cycler to 95^oC
5. Quickly transfer tubes from ice to the temperature cycler and begin thermocycling
6. Typical thermocycle procedure:
   

   Step Name	Temperature	Time
   Denaturation	95 oC	30 seconds
   Cycle Denaturation	95 oC	30 seconds
   Cycle Priming	50-60 oC	60 seconds
   Cycle Extension	72 oC	1 minute per kilobase of target DNA
   Final Extension	72 oC	5 minutes
   Hold Temperature	4 oC	Infinite

7. Repeat the above “cycle” steps 35-45 times.
8. Make sure to keep the samples on ice while you are not using them. The infinite hold sequence allows you the flexibility of leaving your samples in the temperature cycler while you are busy with other lab work.

Notes on this PCR Methodology

• Note 1. It is important to design your PCR primers to be specific to only the regions flanking the target sequence. Typically, specific primers are ~30-40 bases in length.
• Note 2. The Tm (melting temperature) of the primers affect the temperature in Step 3b and the “Cycle priming” step.
• Note 3. For greater accuracy/fidelity while copying DNA, a Pfu polymerase (from Pyrococcus Furiosus) may also be used.
• Note 4. With 35 cycles, the target DNA is amplified 2³⁶ times its initial concentration. For times when you have very little target DNA, you can amplify 45 times or more.
• Note 5. For Primer design, you can use tools such as Primer 3. Tools are also available for calculating the Tm of your primers such as this Tm calculator.
• Note 6. G-C rich sequences will need longer denaturation times (typically up to 5 minutes) because of the additional hydrogen bonding. Any template with more than 60% G-C bases is considered G-C rich.

Applications of PCR found on SciGine

• Bacillus anthracis pXO2 plasmid PCR amplification
• PCR amplification of D-amino-acid oxidase cDNA fragment
• Isolation of gp91 homologue sequences from maize
• Semi-nested RT-PCR amplification of picornavirus
• PCR-RFLP for immunoglobulin receptor polymorphisms

References

• Technique Overview from the National LCDC
• PCR Technology Article from Pubmed
• Research Techniques made simple
• New England Biolabs Protocol
• Integrated DNA Technologies Methodologies