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1
Application of Nucleic Acid Amplification in ClinicalMicrobiology
Gorm Lisby1. IntroductionSince the discovery of the doublehehx structure of DNA (I), no single eventhas had the same impact on the field of molecular biology as the rediscoveryby Kary
Mullls
in the early 1980s of the polymerase cham reaction (PCR) (2-41, which was first published in principle by Keld Kleppe m 1971 (5). Thiselegant technology with its apparent simple theory has revolutronized almostevery aspect of classical molecular biology, and is at the present momentbeginning to make a major impact upon many medical-especrally dtagnostrc-specialities. The field of climcal mrcrobtology has been among the first toembrace the polymerase chain reaction technology, and the expectations of thefuture impact of this technology are high. Fnst and foremost, the diagnosticpossibilities of this technology are stunning, but in this era of emergmg tmple-mentation, rt is crucial to focus not only on the possibrhties, but also on thepitfalls of the technology. Failure to do so will increase the cost of implemen-tation manifold, and ~111risk to disrepute the technology in the eyes of theclinicians.2. PCR-Theory and Problems
2.1.
“Classic”
PCR
2. I. 7. The Principle of Exponential AmplificationThe hallmark of the polymerase chain reaction is an exponential amplifica-tion of a target DNA sequence. Each round of amplificatron IS achieved byannealmg specific oligonucleotides to each of the two complementary DNA
From Methods m Molecular Bfology, Vol 92. PCR m BoanalysrsEdlted by S J Meltzer 0 Humana Press Inc , Totowa, NJ1
 
2
2nd
Fig. 1. The first three cycles of a standard PCR. The tentative annealmg tempera-ture of 55°C needs to be optlmlzed for each PCR set-up
strands after denaturation. Following annealing of the two oligonucleotides
(primers), a thermostable DNA polymerase (6) ~111 produce doublestranded
DNA, thus in theory doubling the amount of specific DNA in each round (Fig.1). After the third round of amplification, a specific product consisting of thetarget DNA fragment between the two primer annealing sites (and including
 
Application of Nucleic Acid Amplification3the two sites) will start to accumulate. When the usual 30-40 rounds of amph-fication are completed, up to several hundred million fold amplification of thespecific target sequence can be achieved. The amplified products can bedetected by numerous methods that vary in sensitivity, accuracy, and feasibil-ity for routine application: From the classical agarose gel electrophoresrs,Southern blot and Sanger dideoxy sequencing to probe capture and visualiza-tion in microtiterplates and direct realtime detection of the product in the PCRtube by fluorescens (7).2.1.2. Primer Selection and Primer AnnealingSeveral aspects must be considered when a primerset for PCR is designed(8). Computer software programs have been constructed to deal with this prob-lem
(9-ll),
and these programs are based on the same considerations, as onehas to take during a “manual” primer design:The primers are typically between 15 and 30 bases long and do not have tobe exactly the same size. However, it is crucial that the melting temperatures ofthe two primer/template duplexes are identical within l-2°C. Since abillion-fold surplus of primers may exist in the beginning of a PCR when com-pared to the target sequence to be amplified, the optimal primer annealing tem-perature of a primer may be higher than the calculated melting temperature ofthe primer/template duplex (where 50% of the DNA molecules are double-stranded and 50% are singlestranded). Several formulas to calculate theannealing temperature exist (12-14), but eventually one has to establish theactual optimal annealing temperature by testing.The location of the target sequence and thereby the size of the amphfiedproduct is not crucial for the sensitivity or the specificity of the analysis, andtypically a fragment of 150-800 bases s amplified. Amplification of productssizing up to 42,000 basepan-s has been reported (15). However, when frag-ments above 1000-2000 basepairs are amplified, problems with templatereannealing can be encountered (15-I 7). The annealing step m a “long-range”PCR is thus a balance between keeping the templates denatured and facilitat-ing primer annealing.The composition of the two primer sequences must ensure specific anneal-ing to the target sequence alone. The probability of this specificity can be madethrough a search in the computer databases (GeneBank or EMBL), but eventu-ally this also has to be established empirically (absence of signals from DNAfrom other microorganisms than the target organism). It is of utmost impor-tance, that the sequences at the 3’ end of the two primers are not homologous,otherwise the two primers will self anneal with primer-dimer products and apossible false negative analysis as result. At the 5’ end of a specific primer, a“tail” consisting of a recognition sequence for a restriction enzyme, a captur-
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frankkrzal

Fould I votre emplacement dans MSN.

reply10 / 21 / 2009
delsiebernhardt

U hebt een hoogste inkepingsplaats

reply10 / 21 / 2009
claudineullmann

keep this stuff coming woot!

reply10 / 21 / 2009
laquandabutay

my wife loves this stuff, i dont

reply10 / 19 / 2009
simonefucillo

je l'ai trouvE tres utile en effet,

reply10 / 18 / 2009
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