文档介绍：M E T H O D S I N M O L E C U L A R M E D I C I N E TM
Molecular
Diagnosis of
ic Diseases
Second Edition
EditedEdited byby
RobRob Elles,Elles, PhDPhD
RogerRoger Mountford,Mountford,
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Optimizing PCR for Clinical Diagnosis
Michael P. Bulman
1. Introduction
The polymerase chain reaction (PCR) has rapidly e an essential tool
within the diagnostic laboratory. Therefore, it is crucial when setting up a new
PCR-based test to ensure that the PCR reaction is carefully designed to be as
robust and reliable as possible. Usually, little optimization is required. However,
there are some instances when a particular region of DNA proves difficult to
amplify by PCR. A number of factors are important to consider when choos-
ing PCR conditions, and these are discussed in this chapter.
2. Materials
Analytical-grade reagents should be used at all stages, unless otherwise
stated.
. PCR Reaction Buffers
As an alternative to the buffer supplied by the manufacturer, use of either of
the following buffers may be beneficial in the PCR.
. Buffer “A” 10X
1. 670 mM Tris-HCl (pH at 25°C).
2. 166 mM ammonium sulfate.
3. 37 mM magnesium chloride.
4. 67 µM etraacetic acid (EDTA).
5. mg/mL bovine serum albumin (BSA).
Filter-sterilize and store in 1-mL aliquots at –20°C.
This is a particularly robust PCR buffer, which provides a good yield of prod-
uct, and is excellent regardless of the quality of the DNA template.
From: Methods in Molecular Medicine, vol. 92: Molecular Diagnosis of ic Diseases, Second Edition
Edited by: R. Elles and R. Mountford © Humana Press Inc., Totowa, NJ
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2 Bulman
. Buffer “R” 20X
1. M Tris-HCl (pH at 25°C).
2. 400 mM ammonium sulfate.
3. 30 mM magnesium chloride.
Filter-sterilize and store in 1-mL aliquots at –20°C.
This buffer is useful for templates that are difficult to amplify,