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Klaus Zimmermann and Josef ences 16–18, 21, 31, 32 and 84). This plates, different PCR strategies and
W. Mannhalter assay is based on competitive co-ampli- modes of detecting PCR products.
Immuno AG fication of a specific target sequence to-
gether with known concentrations of an
Vienna, Austria internal standard in one reaction tube. CONSTRUCTION OF INTERNAL
The internal standard has to share STANDARDS FOR COMPETITIVE
primer recognition sites with the spe- PCR
cific template, both specific template
INTRODUCTION and internal standard must be PCR-am- Generation and testing of suitable
plified with the same efficiency and it internal standards and the choice of
The polymerase chain reaction must be possible to analyze the PCR- primer pairs are among the most crucial
(PCR), first described by Saiki et al. amplified products of specific template and time-consuming aspects of setting
(79), is a highly sensitive and specific and internal standard separately. Quan- up a competitive PCR protocol. The
methodology for detection of nucleic titation is then performed by comparing simplest way to choose suitable pri-
acids and a useful tool for quantitation the PCR signal of the specific template mers is to use, as far as possible,
of the amount of specific nucleic acids with the PCR signals obtained with primers already widely used and/or
present in a sample. The simplest ap- known concentrations of the competitor well described. If new primers have to
proach to quantitation of PCR and re- (the internal standard). Ever since this be designed, computer programs in
verse transcription PCR (RT-PCR) method was first described (6,38,99), it combination with sequence data bases
products (reviewed by References 17, has been widely used for quantitation may be helpful tools.
21, 31 and 32) is measurement of the of cellular RNA and DNA as well as vi- General concepts of PCR primer de-
amount of amplification product in the ral and bacterial nucleic acids. Exam- sign have been reviewed by Dieffen-
exponential phase by reference to the ples reported on include the quantita- bach et al. (26). Since different primer
dilution series of an external standard. tion of cytokine expression (6,38,52,91, pairs for the same gene can exhibit up
However, accurate quantitation with 99,103) and of viral nucleic acids such to 1000-fold differences in sensitivity
this type of PCR is hampered by a as hepatitis B (46), hepatitis C (9,40,53, (44), special emphasis should be placed
number of variabilities that can occur 58,75,78,106), human cytomegalovirus on testing the specificity and efficiency
during sample preparation or in the (35), herpes simplex virus (74) or hu- of primer pairs in advance before inter-
course of the reaction, and minor varia- man immunodeficiency virus type 1 nal standards are generated. Once the
tions in reaction conditions are greatly (HIV-1) (3,5,36,51,62,70,71,81,88,92) appropriate primer pair has been select-
magnified during the amplification and quantitation of bacteria, especially ed, various strategies for the construc-
process. These variabilities may partly of slow-growing species such as my- tion of internal standards may be used.
be overcome by normalizing the cobacteria (55). Furthermore, the utili- Internal standards for competitive
amount of PCR products of the specific ty of competitive PCR or RT-PCR has PCR or RT-PCR are DNA or RNA
template with respect to an internal ref- been demonstrated in the quantitation fragments sharing the primer recogni-
erence template such as the cellular of mitochondrial DNA (100) or mRNA tion sequences with the specific target
gene β-globin (20) amplified in the expression (29,69) and assessment of yet yielding PCR products that are dis-
same reaction tube. hereditary deficiencies (e.g., Reference tinguishable from the wild-type tem-
Alternatively, limiting dilution using 43) or leukemias (22,93). plate. The easiest way to distinguish
a nested primer methodology (57,90) Considering the hundreds of pub- between wild-type template and inter-
can be used in combination with Pois- lished papers on the use of competitive nal standard is by differences in the size
son statistics for evaluation of the PCR, it is not surprising that a great va- of the two products. This can be
results. riety of protocols exist. In the present achieved, for example, by constructing
The most precise quantitation of article, we shall review this methodolo- standards having the same sequence as
DNA and RNA can, however, be ob- gy, concentrating in particular on tech- the specific target but containing a dele-
tained by competitive PCR and com- nical aspects of competitive PCR, such tion or an insertion. The simplest con-
petitive RT-PCR (reviewed in Refer- as the construction of competitive tem- struction procedure is to use a compos-