PCR Process Steps Explained - Cole-Parmer

Home Technical Resource Library + SAVE TO MY DOCS
Articles and White Papers

 PRINT
Polymerase Chain Reaction (PCR) Process Steps
Polymerase Chain Reaction (PCR) Process Steps

AVAILABLE IN: English
Studying isolated pieces of DNA is nearly impossible. Large amounts of a sample of DNA are necessary for molecular and
genetic analyses. Sometimes called molecular photocopying, conventional polymerase chain reaction (PCR) is a technique
used to amplify (replicate) trace amounts of DNA and RNA from a sample. A PCR thermal cycler is used to produce the
large amounts required for research.
The PCR process can be used for a wide variety of laboratory and clinical applications and purposes. Forensic labs use it to
analyze DNA samples from a crime scene. Clinical health care labs use it to diagnose patients infected from a virus.
Pharmaceutical research labs use it to analyze and duplicate DNA and RNA samples for use in the manufacturing of drugs
and vaccines.
Sample Collection and Preparation for PCR

Four components (reagents or chemicals) are needed for the PCR process:
A DNA or RNA sample (from saliva, blood, hair, skin scraping, etc.)
DNA primers: short single-stranded DNA that promotes synthesis of a complementary strand of nucleotides
DNA polymerase: an enzyme that aids in the synthesis of a complementary strand of DNA
Nucleotide solution mix containing adenine (A), thymidine (T), cytosine (C), and guanine (G) used to build duplicate DNA strands
The PCR Steps Explained

The PCR process begins with a segment of a DNA sample placed in a suitable tube along with the reagents and chemicals
listed above. The tube is placed into the PCR machine or thermal cycler. The thermal cycler takes the solution through a 3-
step process: denaturation, annealing, and extension.
Step 1 - Denaturation
The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The heat breaks the hydrogen
bonds of the original DNA sample and separates the DNA into single strands (this is termed denaturation of double-
stranded DNA).
Step 2 - Annealing
The sample mixture is then cooled to between 50 to 60°C (122 to 140°F) allowing the DNA primers and the DNA
polymerase enzyme to bind to the individual strands of DNA that were separated by the heat (this is termed annealing of
the primers). At this point, the nucleotides (A, T, C, G) from the added mixture solution will pair with the individual
separated strands of DNA that resulted from the heating process.
Step 3 - Extension
Once joined together, they form a new complementary strand of DNA (termed extension of the DNA). Thus, a new
duplicate double-stranded DNA molecule has been formed from each of the single strands of the original sample molecule.
The cycle is then repeated about 35 to 40 times using the thermal cycler which automatically repeats the heating and
cooling cycles of the process. Resulting DNA sequence is doubled each time the heating/cooling cycle is conducted by the
cycler. Thus, what started out as a single short segment of DNA from one sample can be amplified to form millions of
copies after 35 doubling cycles.
Courtesy: National Human Genome Research Institute

Step 4 - Analysis with Electrophoresis
Once the PCR process is completed, the resulting amplified (replicated) segments generated can then be compared to other
nucleotide segments from a known source. The PCR-generated nucleotide sequences are then placed next to known
nucleotide sequences from humans, pathogens, or other sources in a separating gel. Electrical current is then run through
the gel, and the various nucleotide sequences within the gel form bands that resemble a ladder, according to their electrical
charge and molecular size. This is termed gel electrophoresis. Bands or ladder-like steps that migrate to the same levels in
the gel show identity of nucleotide sequences. This method is one of the most popular ways PCR tests are completed.
qPCR
qPCR is a variation of conventional PCR and allows analysis of the amplified/replicated DNA in real-time during the usual
40 cycles of the procedure using fluorescent dyes. The fluorescent dyes attach to some of the nucleotide strands allowing
users to measure specific products and their amounts during the amplification/replication cycles. Special thermal cyclers,
known as real-time thermal cyclers, are used for qPCR. As well as heating and cooling the tubes containing the PCR
reagents, they can measure the fluorescence inside the tube at every cycle. This allows users to skip the gel electrophoresis
or other secondary procedures needed for final analysis of the PCR products, thus producing more rapid results.
Browse PCR and qPCR products and equipment available from Cole-Parmer
Last Updated: 05/21/20
Cole-Parmer India Pvt. Ltd. | 403, A-Wing, Delphi, Hiranandani Business Park Powai, Mumbai,
400 076 India
Telephone: 91-22-61394444 , 1-800-266-1244 | Fax: 91-22-61394422 | Email: [email protected]
© 2021 Cole-Parmer Instrument Company, LLC. All Rights Reserved.

PCR Process Steps Explained - Cole-Parmer

Uploaded by

Copyright:

Available Formats

PCR Process Steps Explained - Cole-Parmer

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

PCR Process Steps Explained - Cole-Parmer

Uploaded by

Copyright:

Available Formats

Home Technical Resource Library + SAVE TO MY DOCS

Articles and White Papers

Polymerase Chain Reaction (PCR) Process Steps

Sample Collection and Preparation for PCR

The PCR Steps Explained

Courtesy: National Human Genome Research Institute

Last Updated: 05/21/20

You might also like