In part 1.1, of the Biotech tropicana Inc PCR
protocol publication series, we discuss the basic infrastructure requirements
of a PCR laboratory. Here we describe variants of PCR laboratory and highlight
key organizational concepts that must be taken into consideration in setting up
a PCR laboratory.
Keywords:PCR; PCR facility; Resource-poor Settings; Alternative Technologies
Background:
PCR
technology has become a necessary component of any modern molecular biology
laboratory. The PCR is a very powerful method that permits amplification of
trace amount of DNA to a practically limitless quantity. However, the
technology has its weaknesses. PCR is very susceptible to contamination by its
own products. Contamination avoidance is the most important factor to be
considered in setting up a PCR lab. Amplicons escaped from previous PCR
experiments are a major source of contamination of a PCR laboratory. PCR
systems may also be contaminated from a variety of sources including the DNA
template itself and, previous amplification of plasmid. [1;2]
In part 1.1
we discussed the basic requirements of a PCR laboratory at two levels, 1)
general requirement of a chemistry laboratory facility and, 2) general
requirement of a PCR facility. Here we must emphasize that at Biotech
tropicana, Inc, we make a distinction between the terms "PCR laboratory” and "PCR
facility”. By "PCR facility” we, basically referred to the building in which
the PCR laboratory is housed and basic structural components of the house such
as doors, laboratory bench and, other non removable components. We construe the
term PCR laboratory to encompass PCR specific equipments such as PCR execution
protocols, thermocyclers, and post PCR analysis components such as gel
electrophoresis equipments. Consistent with our objectives to achieve the
highest possible simplification of the PCR concept, we will maintain the
distinction between the two terms through out this work.
Here we
introduce additional basic concepts applicable to all PCR facilities to include
structural organization of the facility to permit a) linear and unidirectional
workflow, b) task specific dedication of equipments, c) color coding of
equipments. We then explain the basis of variations in PCR facility
organization as supported by selective case studies.
Description:
To avoid
contamination and obtain high quality PCR results, linearity is an important
concept in establishing a PCR facility. The facility must be designed to permit
unidirectional work flow from pre-PCR room to post-PCR room. Within each room,
substructures of the room must also be organized to permit unidirectional
protocol execution. The linear and unidirectional organization concepts are well
illustrated by Ahmed E. Yousef in [3, page 7]
Equipment
must be dedicated to room and within rooms equipments must dedicated to the
particular task they are intended for. According to Mifflin T, even
communication tools such as computers, and telephones must be dedicated. [1]
Color
coding of equipments some equipments such pipettes may help maintain the
dedication requirements by avoiding confusions during test executions. Colored
codes will mark off each pipette
dedicated to a particular task.
There
are variations from the standard "two” rooms, pre-PCR and post-PCR,
organization. The basis of the variations may be motivated by purpose of the
experiments, and technical aspects of equipments. Compared Mifflin, T at page 8
[1]proposing
a two rooms organization with Hussein at page 2 [2] , proposing a basic three rooms facility. According to
Hussein A, specimen preparation occurred in the first area. There again
specimen may vary, thereby increasing variation in Hussein, A, first work area
concept. In practice variations in specimen preparation is limitless; and
depend on the application purpose of the scientist directing the establishment
of the facility. [1]Since the development of the PCR technology in the mid
1980, automations have been introduced at each of the four steps, of the PCR
execution protocols. Automation tends to reduce the number of equipments and
the number of rooms required for an optimal PCR application. Real Time PCR
eliminates the need for post-PCR analysis by reading the result of the PCR
amplification in real time, thereby eliminating the needs for a post-PCR room. [1]
Discussion
In part1.1,
we discuss the basic requirements of a PCR facility. In the current part1.2, we
further expand the basic concept applicable to establishing a contamination
free PCR facility. We discuss the concept of linearity and unidirectional PCR
test execution, dedication of equipments and variations in facility design.
Part1.3 will discuss basic equipments in a PCR lab for an optimal contamination
free PCR application. The attached pictures show variations in PCR facilities.
We simulate each version using our card bard models. Available at http://btitechtrials.ucoz.com/photo, PCR Labs
Conclusion :
Part1.2 of the Biotech tropicana, Inc PCR
protocol tailored to the resource-poor settings introduced the concepts of
linearity, unidirectional, dedication and color coding of equipments, and
variations in the design of a PCR facility.
[3]Nathan Associates Inc, Ahmed
E. Yousef,Assisting GOEIC to establish
a PCR Laboratory, (Technical Report). Available at http://pdf.usaid.gov/pdf_docs/PNADJ772.pdf.Accessed
January 18, 2010
