Introduction to PCR

Polymerase chain reaction, or PCR, is a tool used in many biotechnology applications that makes it possible to study small, specific fragments of DNA information at the gene level. The primary activity during PCR is the copying and recopying of a short DNA sequence to create millions of identical DNA fragments. The DNA fragments can then be further characterized and/or checked for purity by running them in an agarose or polyacrylamide gel system. In some cases a southern blot is subsequently performed as well. In short, PCR is DNA synthesis in a tube.

Applications for PCR

PCR is routinely used in a variety of laboratories in both the public and private sectors. Some common applications of PCR include:

• Medical diagnostics
• Gene / protein studies
• Therapeutic drug design
• Paternity / maternity identification
• Forensics
• Missing persons
• Evolutionary studies
• Endangered species identification and protection

Step by Step PCR and Common PCR Reagents

While the optimal PCR protocol may be different for various DNA fragments based on the size and deoxynucleotide composition of the selected fragment, the theory and sequence behind PCR reactions does not change. Below you will find a brief description of the various protocol steps and common reagents that are necessary for PCR to effectively produce DNA replicates.

Step by Step PCR

1. DNA fragment of choice is placed in a tube with the reagents necessary to perform PCR.
2. A short segment of DNA is unzipped (denatured) by breaking the H bonds with extreme heat (approximately 95ºC), creating two DNA templates.
3. One end of each DNA template is recognized by forward and reverse primers and the primers are annealed to the DNA by lowering the temperature (35ºC - 65ºC).
4. DNA polymerase molecules create copies of each template starting at the end of each primer, producing two new fragments called extensions. This is performed at approximately 72ºC.
5. The replication process (step 2) is repeated 25 – 35 times for each newly synthesized fragment, causing the number of identical fragments to increase exponentially with each cycle.
6. One double stranded fragment of DNA that undergoes 35 cycles of PCR results in over 34 billion (235) fragments in approximately 3 hours!

Example of a PCR Protocol

Common reagents used for PCR reactions

All PCR protocols need the same basic reagents to drive each step of the PCR cycle. Specialized formulations can be obtained from many sources for specific, advanced research needs and optimization. Click on the reagents below for ordering information through WARD’S.

• Taq polymerase – special DNA polymerase (enzyme) that is able to remain active at high temperatures.
• dNTPs – The 4 deoxynucleotides that make up DNA (A, C, T, G).
• Magnesium Chloride (MgCl2) – enzyme cofactor needed for polymerase activity.
• PCR Master Mix – premixed solution of Taq polymerase, dNTPs and magnesium chloride at optimal concentrations.

Optimizing PCR Protocols

Various fragments or sequences of DNA have different physical and chemical characteristics that can affect the rates and effectiveness of the activities that take place during PCR. Determining the most appropriate PCR conditions for a particular fragment of DNA may include optimizing the following:

• Temperature and time to activate Taq polymerase
• Temperature and time to allow primer annealing
• Temperature and time for replication
• Concentration of reagents, especially primers, dNTPs, and MgCl2)
• Number of replication cycles

Processes and reagents used to extract DNA and purify DNA fragments may also contaminate the DNA sample and inhibit replication. Some common replication inhibitors include:

• DNase
• RNase
• EDTA
• Alcohol
• DNA fragments from previous experiments

Using Thermocyclers for PCR

Simply stated a thermocycler is a heating block that is regulated by a computer. Thermocyclers allow the scientist or technician to “walk away” as the PCR cycles are performed as well as maintain tight control over the temperatures needed to complete the PCR.

In the classroom the use of a thermocycler allows students to gain the valuable experience of running a PCR reaction with a piece of equipment that is used daily throughout biotech and pharmaceutical research facilities. It also frees up time for other activities since thermocyclers are programmable and can even be set to maintain a “storage” temperature for several hours.

Shopping for Thermocyclers

There are many models of thermocylers available from several manufacturers, and choosing the right model for your educational needs may seem like a bit intimidating.

Some of the feature that should be considered when comparing models includes:

• Storage capacity for preprogrammed cycles
• Communication / connectivity options for personal computers and networks
• Maximum number of samples that can be run per activity
• Maximum number of PCR protocols that can be run at once (gradient feature)
• Temperature range
• Ramp rate (rate of temperature change)
• Weight and dimensions
• Warranty
• Heated or unheated lid
• Screen display characteristics

Ward’s Natural Science Featured Thermocyclers

EdvoCycler by Edvotek
The all-new EdvoCycler brings affordable PCR to the classroom without compromise. The 0.2 ml tube block has room for up to 25 students’ samples and comes pre-programmed with EDVOTEK PCR protocols. These programs may be modified or deleted, and there is extra memory for more. The vivid 7 line LCD displays all program parameters simultaneously without any scrolling. A heated oil-free lid makes operation a snap. Proudly made in the USA and backed by a 2-year warranty!

EdvoCycler Features:

• 25 x 0.2 ml Tube Block
• Heated Oil-Free Lid
• Vivid 7 Line LCD Display
• Pre-Programmed, Changeable PCR Protocols
• Temperature Range: 10°C–99°C
• Maximum Ramp Rate: 2°C/sec
• Weight & Dimensions: 11 lbs. & 16″ x 8.5″ x 7″
• 2 Year Warranty
• Made in USA

Teche Endurance TC-312 by Techne
With its user-friendly programming, heated lid, easy-to-read LCD, and low price, this compact, remarkably fast thermal cycler is ideal for school labs, but is reliable enough to stand up to the demands of a molecular scientist. It automatically provides ideal temperature conditions for PCR DNA amplification reactions. It also features memory that stores up to 80 programs, parallel printer port, and an RS232 port to print profile information that was programmed for the run. You can also program this cycler with PCR analysis software that you can download from the manufacturer’s web site.

Teche TC-312 Features:

• 25 x 0.2 ml Tube Block
• Heated Oil-Free Lid
• Easy to Read LCD Display
• Temperature Range: 4°C–99°C
• Maximum Ramp Rate: 2.0°C/sec
• Dimensions: 13″ x 6.75″ x 7.5″
• 80 Program Storage Capacity
• 4 Year, 80,000 Cycle Warranty

MultiGene II by Geneq
Featuring a heated lid for oil-free cycling, this fast, uniform cycling unit offers user friendly operation. Heating and cooling are accomplished electronically by Peltier units. The unit itself is programmed to control the heating and cooling with an algorithm that accurately simulates sample temperature. Programs can be run immediately upon entering, or up to 99 programs can be saved to the memory for later use. The built-in software also allows for successive time and temperature increments and decrements, end of cycling elongation steps, and extended period soaks at 4°C. The cycler can also be used for holding specific temperatures, allowing it to take the place of a traditional water bath, heat block or incubator. Includes a user’s manual.

MultiGene II Features:

• 25 x 0.2 ml Tube Block
• Heated Oil-Free Lid
• 40 character LCD Display
• 99 Program Storage Capacity
• Temperature Range: 4°C–99.9°C
• Maximum Ramp Rate: 2.2°C/sec
• Dimensions: 8.5″ x 11.25″ x 7.0″

More Related Products

Find equipment, activities and other related supplies.

• WARD’S Biotech category
• WARD’S PCR category

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